Heit Lab Wiki - User contributions [en]

Immunostaining

2024-09-17T11:37:24Z

Admin: /* PEM Buffer: */

This procedure was optimized using the 4G10 (anti-phosphotyrosine) antibody, but should work well for many other antibodies. For a generic protocol, add an additional 20-60min at RT fixing step after the 4C fixing step. New fixative is not needed, simply move the plate to the bench & cover with foil. Antibody concentrations will need to be optimized for each antibody.  Please enter details for all optimized antibodies in the table at the end of the protocol. 

 

== Protocol ==

#Fix cells in 4% PFA/PBS for 20' at room temperature. [https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC4958280/ To preserve membrane structures], fix with 4% PFA in PEM, 37C, 10 min.
#Wash 3X PBS
#Permeabilize and block using antibody buffer, 1hr at RT.
#Add primary antibody at desired concentration in antibody buffer, 20C for 1 hour or 4C overnight.
#*Use hanging drop method:
#**Put 100ul antibody mixture on a piece of parafilm
#**Flip coverslip onto drop
#**Cover with foil or box and leave undisturbed for 20 min (surface staining) or up to overnight (intracellular staining; minimum 1 hr). '''KEEP LEVEL'''.
#Was 3X 15min with PBS.
#Add secondary antibody at desired concentration (typically 1:500 to 1:1000) in antibody buffer:
#*Use hanging drop method:
#**Cover with foil and leave undisturbed for 1-2 hours 
#Wash 3X 15min in PBS.

Cell can be imaged in PBS, or mounted using DAKO on a slide. Imaging is generally better in PBS, as DAKO can distort cells as it dries.

 

== Recipes ==

===== PEM Buffer: =====

* 80 mM PIPES pH 6.8 (2.419g/100 mL)
* 5 mM EGTA (190 mg/100 mL)
* 2 mM MgCl2 (19 mg/100 mL)

===== Antibody Buffer: =====

PBS + 0.1% triton X-100 + 5% donkey serum or 2.5% BSA (use goat serum of secondary is derived in goats).

''For 10ml:''

*9.5ml PBS
*10ul Triton X-100 (exclude if surface-staining)
*500ul serum or 250mg BSA

 

Note: 1% BSA can be used in place of serum in many cases, especially after step 3.

 

== Working Conditions ==

{| width="100%" cellspacing="1" cellpadding="1" border="1"
|-
| '''Antibody '''
| '''Fixation''' 
| '''Primary Conditions '''
| '''Washes''' 
| '''Secondary Conditions''' 
| '''Washes '''
|-
| 4G10 
| 4% PFA, 20', 4C 
| 1:100 in antibody buffer, overnight, 4C 
| 3 x 15min PBS 
| 1:200 anti-mouse in antibody buffer, 1 to 2 hours, room temp. 
| 3 x 15min PBS 
|-
| 4G10 
| 4% PFA, 20', 4C 
| 1:100 in antibody buffer, 4hrs, 20C 
| 3 x 15min PBS 
| 1:200 anti-mouse in antibody buffer, 1 to 2 hours, room temp. 
| 3 x 15min PBS 
|-
| THE His6 Antibody ([http://www.genscript.com/antibody/A00186-THE_sup_TM_sup_His_Tag_Antibody_mAb_Mouse.html GenScript A00186]) 
| 4% PFA, 20', 4C 
| 1:250-1:500 in antibody buffer, 1-4hrs, 20C 
| 3 x 15min PBS 
| 1:1000 anti-mouse in antibody buffer, 1 to 2 hours, room temp. 
| 3 x 15min PBS 
|}

THP1 Culture and Differentiation

2024-08-21T17:49:38Z

Admin: /* Protocol */

==== Maintenance ====
THP1 cells ([https://www.atcc.org/products/tib-202?matchtype=&network=x&device=c&adposition=&keyword=&gad_source=1&gclid=Cj0KCQjw-5y1BhC-ARIsAAM_oKlZTKs5zplX22cd1pVZB7E0-Cq7fDL5pcruuijrK0PqV9a-5LG5l24aAoEqEALw_wcB ATCC TIB-202]) should be maintained in RPMI + 10% FBS, with cells split 1:5 into fresh medium when the cell density reaches 1 x 106 cells/mL. Do not allow cell density to exceed 1.5 x 106 cells/mL.
----

==== '''Classical Macrophage Differentiation''' ====
# Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
# Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
# Add sufficient medium to bring the well volume up to 1 mL.
# Add 10 uL of 100 ng/mL PMA (final concentration of 100 ng/mL), culture for 3-5 days.

----

==== Subtype-Specific Macrophage Differentiation ====

===== Differentiation Medium =====

* 10 mL of RPMI + 10% FBS
* 1.0 uL of 100 uM PMA

===== Protocol =====

#Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
# Add 200 uL of THP1 cells, aiming for 150,000 to 200,000 undifferentiated THP1 cells in each well.
# Add 1 mL of M0 differentiation medium (final PMA concentration at 1.2 mL is 5 ng/mL).
# Culture for 24 hours at 37C/5% CO2.

====== M0 Differentiation ======

# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PBS.
# Replace medium with RPMI + 10% FBS (no PMA).
# Culture an additional 48 hours.

====== M1 Differentiation ======

# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PBS.
# Replace medium with RPMI + 10% FBS (no PMA) + 1 μL/mL LPS + 1 μL/mL INFγ.
# Culture an additional 48-72 hours.

====== M2 Differentiation ======

# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PBS.
# Replace medium with RPMI + 10% FBS (no PMA) + 2 μL/mL IL-4
# Culture an additional 48-72 hours.

----

==== Dendritic Cell Differentiation ====
Protocol from [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866978/ PMID 36674966]

===== Media Preparation =====

====== iDC Medium ======
* 10 mL RPMI + 10% FBS
* 35 μL 2-mercaptoethanol
* 100 μL of 100X antibiotic/antimycotic
* 5 μL of GM-CSF (20 μg/mL stock, 100 ng/mL final)
* 10 μL of IL-4 (10 μg/mL stock, 100 ng/mL final)

====== mDC Medium ======

* 10 mL RPMI + 10% FBS
* 35 μL 2-mercaptoethanol
* 100 μL of 100X antibiotic/antimycotic
* 5 μL of GM-CSF (20 μg/mL stock, 100 ng/mL final)
* 20 μL of IL-4 (10 μg/mL stock, 200 ng/mL final)
* 20 ng/mL TNFα
* 200 ng/mL ionomycuin

====== ''iDC Procedure'' ======

# Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
# Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
# Add 1 mL of iDC medium to each well, incubate 3 days.
# Replace medium and incubate an additional 2 days.

====== mDC Procedure ======
''Starting with iDCs from above''

# Replace medium with mDC medium.
# Incubate 2 days.

THP1 Culture and Differentiation

2024-08-21T14:58:50Z

Admin: /* Subtype-Specific Macrophage Differentiation */

==== Maintenance ====
THP1 cells ([https://www.atcc.org/products/tib-202?matchtype=&network=x&device=c&adposition=&keyword=&gad_source=1&gclid=Cj0KCQjw-5y1BhC-ARIsAAM_oKlZTKs5zplX22cd1pVZB7E0-Cq7fDL5pcruuijrK0PqV9a-5LG5l24aAoEqEALw_wcB ATCC TIB-202]) should be maintained in RPMI + 10% FBS, with cells split 1:5 into fresh medium when the cell density reaches 1 x 106 cells/mL. Do not allow cell density to exceed 1.5 x 106 cells/mL.
----

==== '''Classical Macrophage Differentiation''' ====
# Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
# Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
# Add sufficient medium to bring the well volume up to 1 mL.
# Add 10 uL of 100 ng/mL PMA (final concentration of 100 ng/mL), culture for 3-5 days.

----

==== Subtype-Specific Macrophage Differentiation ====

===== Differentiation Medium =====

* 10 mL of RPMI + 10% FBS
* 1.0 uL of 100 uM PMA

===== Protocol =====

#Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
# Add 200 uL of THP1 cells, aiming for 150,000 to 200,000 undifferentiated THP1 cells in each well.
# Add 1 mL of M0 differentiation medium (final PMA concentration at 1.2 mL is 5 ng/mL).
# Culture for 24 hours at 37C/5% CO2.

====== M0 Differentiation ======

# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PMA.
# Replace medium with RPMI + 10% FBS (no PMA).
# Culture an additional 48 hours.

====== M1 Differentiation ======

# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PMA.
# Replace medium with RPMI + 10% FBS (no PMA) + 1 μL/mL LPS + 1 μL/mL INFγ.
# Culture an additional 48-72 hours.

====== M2 Differentiation ======

# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PMA.
# Replace medium with RPMI + 10% FBS (no PMA) + 2 μL/mL IL-4
# Culture an additional 48-72 hours.

----

==== Dendritic Cell Differentiation ====
Protocol from [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866978/ PMID 36674966]

===== Media Preparation =====

====== iDC Medium ======
* 10 mL RPMI + 10% FBS
* 35 μL 2-mercaptoethanol
* 100 μL of 100X antibiotic/antimycotic
* 5 μL of GM-CSF (20 μg/mL stock, 100 ng/mL final)
* 10 μL of IL-4 (10 μg/mL stock, 100 ng/mL final)

====== mDC Medium ======

* 10 mL RPMI + 10% FBS
* 35 μL 2-mercaptoethanol
* 100 μL of 100X antibiotic/antimycotic
* 5 μL of GM-CSF (20 μg/mL stock, 100 ng/mL final)
* 20 μL of IL-4 (10 μg/mL stock, 200 ng/mL final)
* 20 ng/mL TNFα
* 200 ng/mL ionomycuin

====== ''iDC Procedure'' ======

# Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
# Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
# Add 1 mL of iDC medium to each well, incubate 3 days.
# Replace medium and incubate an additional 2 days.

====== mDC Procedure ======
''Starting with iDCs from above''

# Replace medium with mDC medium.
# Incubate 2 days.

THP1 Culture and Differentiation

2024-08-07T15:13:33Z

Admin:

==== Maintenance ====
THP1 cells ([https://www.atcc.org/products/tib-202?matchtype=&network=x&device=c&adposition=&keyword=&gad_source=1&gclid=Cj0KCQjw-5y1BhC-ARIsAAM_oKlZTKs5zplX22cd1pVZB7E0-Cq7fDL5pcruuijrK0PqV9a-5LG5l24aAoEqEALw_wcB ATCC TIB-202]) should be maintained in RPMI + 10% FBS, with cells split 1:5 into fresh medium when the cell density reaches 1 x 106 cells/mL. Do not allow cell density to exceed 1.5 x 106 cells/mL.
----

==== '''Classical Macrophage Differentiation''' ====
# Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
# Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
# Add sufficient medium to bring the well volume up to 1 mL.
# Add 10 uL of 100 ng/mL PMA (final concentration of 100 ng/mL), culture for 3-5 days.

----

==== Subtype-Specific Macrophage Differentiation ====

===== Differentiation Medium =====

* 10 mL of RPMI + 10% FBS
* 10 uL of 100 ng/mL PMA

===== Protocol =====

#Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
# Add 200 uL of THP1 cells, , aiming for 150,000 to 200,000 undifferentiated THP1 cells in each well.
# Add 1 mL of M0 differentiation medium (final PMA concentration of 5 ng/mL).
# Culture for 24 hours at 37C/5% CO2.

====== M0 Differentiation ======

# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PMA.
# Replace medium with RPMI + 10% FBS (no PMA).
# Culture an additional 48 hours.

====== M1 Differentiation ======

# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PMA.
# Replace medium with RPMI + 10% FBS (no PMA) + 1 μL/mL LPS + 1 μL/mL INFγ.
# Culture an additional 48-72 hours.

====== M2 Differentiation ======

# After 24 hr of PMA treatment (e.g. step 4, above), wash the cells 3x with pre-warmed PMA.
# Replace medium with RPMI + 10% FBS (no PMA) + 2 μL/mL IL-4
# Culture an additional 48-72 hours.

----

==== Dendritic Cell Differentiation ====
Protocol from [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866978/ PMID 36674966]

===== Media Preparation =====

====== iDC Medium ======
* 10 mL RPMI + 10% FBS
* 35 μL 2-mercaptoethanol
* 100 μL of 100X antibiotic/antimycotic
* 5 μL of GM-CSF (20 μg/mL stock, 100 ng/mL final)
* 10 μL of IL-4 (10 μg/mL stock, 100 ng/mL final)

====== mDC Medium ======

* 10 mL RPMI + 10% FBS
* 35 μL 2-mercaptoethanol
* 100 μL of 100X antibiotic/antimycotic
* 5 μL of GM-CSF (20 μg/mL stock, 100 ng/mL final)
* 20 μL of IL-4 (10 μg/mL stock, 200 ng/mL final)
* 20 ng/mL TNFα
* 200 ng/mL ionomycuin

====== ''iDC Procedure'' ======

# Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate.
# Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture).
# Add 1 mL of iDC medium to each well, incubate 3 days.
# Replace medium and incubate an additional 2 days.

====== mDC Procedure ======
''Starting with iDCs from above''

# Replace medium with mDC medium.
# Incubate 2 days.

Staining for GSD

2024-08-07T12:40:07Z

Admin: /* GSD Buffer 4 (AlexaFluor Optimized Buffer): */

Ground State Depletion (GSD) microscopy detects fluorophore positions independently of intensity or staining density.  As such, it is critical that proper blocking, fixing and staining methods be used in order to avoid off-target fluorescence.  Moreover, coverslips must be of #1.5 (0.17mm) thickness purchased from vendors who can provide coverslips with no fluorescent particulates.  That, or coverslips must be rigorously cleaned (e.g. [http://en.wikipedia.org/wiki/Piranha_solution Piranha cleaning]).  At this time the best coverslips appear to be [http://www.emsdiasum.com/microscopy/products/histology/coverglasses.aspx EMS #72222-01].  As of this writing, coverslips from VWR and Fisher are not GSD-compatible. 

Note: careful selection of secondary antibody fluorophores is key to success in GSD.  Make sure you are using established GSD-compatible fluorophores.

== Protocol - Fixation ==

Careful fixation is required to stabilize cell structures while preserving antigenicity and fluorescent protein activity. The following fixation protocol generally works well: 

#Fix cells in 4% PFA in PBS.
#*If cytoskeletal structure is critical, fix for 10min at 10-12C, followed by 20 min at room temperature
#*otherwise, fix for 20 min at room temperature 
#Wash 3X PBS, 30 sec/wash.
#Continue staining as follows:
#*If cells were transfected with fluorescent proteins and no other staining is required, store cells in blocking buffer until imaging.
#*If intracellular structures are to be immunostained, follow the instructions for intracellular staining. Use this protocol for both intracellular and mixed intracellular/extracellular staining.
#*If only extracellular structures are to be immunostained, follow the instructions for extracellular staining. 

== Protocol - Intracellular Staining ==

Use this protocol when immunostaining intracellular structures. Use this protocol if simultaneously staining intracellular and extracellular structures. 

#Permeabilize and block using permibilization buffer, 1hr at RT.
#*During permeabilization, prepare primary and secondary antibodies in permeabilization buffer. Antibody concentrations are generally the same as those used for conventional immunofluorescent microscopy.
#Stain with primary antibody, room temperature, for 1 to 1.5 hours:
#*Use hanging drop method:
#**Put 100ul of antibody(s) in permeabilization buffer on a piece of parafilm. A highly elevated drop should form.
#**"Float" coverslip, cell-side-down, on drop.
#**Cover with foil or box and leave undisturbed (KEEP LEVEL).
#Wash 3X 15min with blocking buffer.
#Add secondary antibody(s) in permeabilization buffer. Conventional staining or the hanging drop method can be used
#*Secondary staining should be for 1-2 hours, room temperature.
#Wash 3X 15 min in PBS (no BSA).
#Post-fix with 2% PFA in PBS, 20 min at room temperature
#Wash 3X 30 sec with PBS (no BSA) 

Cells should be maintained in PBS until imaging. For imaging cells need to be either mounted in a depression slide using a GSD-appropriate media (e.g. PBS + 50mM cystamine), or coated in PVA. 

== Protocol - Extracellular Staining ==

This protocol should be used only for staining extracellular structures. If staining both extracellular and intracellular structures, pool the antibodies and follow the intracellular staining protocol, above.

#Block the sample for 1 to 1.5 hours with 5% BSA in PBS
#Stain with primary antibody, room temperature, for 1 to 1.5 hours:
#*Use hanging drop method:
#**Put 100ul of antibody(s) in permeabilization buffer on a piece of parafilm. A highly elevated drop should form.
#**"Float" coverslip, cell-side-down, on drop.
#**Cover with foil or box and leave undisturbed (KEEP LEVEL).
#Wash 3X 15 min with PBS + 5% BSA.
#Add secondary antibody(s) in permeabilization buffer. Conventional staining or the hanging drop method can be used
#*Secondary staining should be for 1-2 hours, room temperature.
#Wash 3X 15 min in PBS (no BSA).
#Post-fix with 2% PFA in PBS, 20 min at room temperature
#Wash 3X 30 sec with PBS + 5% BSA. 

 

== Fluorophore & Fluorescent Protein Selection ==

{| width="80%" cellspacing="1" cellpadding="1" border="1"
|-
| '''Fluorophore''' 
| '''Excitation '''
| '''Emission''' 
| '''Notes '''
|-
| Cy3 
| 550 
| 570 
| Slow to deplete, but blinks forever. Orange/Red emission. 
|-
| Dylight 649 
| 650 
| 670 
| Best tested fluorophore to-date. Far-Red emission. 
|-
| YFP 
| 514 
| 527 
| Only confirmed GSD-compatible fluorescent protein at this time. Cannot be combined with orange/red emitting fluorophores 
|-
| Alexa 488
| 488
| 505
| Only good green fluorophore identified to date. Slow to deplete but blinks well.
|-
|Alexa 555
|555
|565
|Equivalent in performance to Cy3, perhaps with slightly better photostability
|-
|Alexa 647
|647
|671
|Similar to Dylight 649, but faster depleating. Blinks forever.
|}

 

== Sample Preparation Buffers ==

===== 4% PFA =====
1 mL of fixative is required for each well of a 12-well plate, or 1.5 mL per well of a 12-well plate. For 4 mL of PFA mix:
*400 ul of 10x PBS
*1 ml of 16% PFA
*2.6 ml of ddH2O
 

===== 2% PFA =====

1 mL of fixative is required for each well of a 12-well plate, or 1.5 mL per well of a 12-well plate. For 4 mL of PFA mix:

*400 ul of 10x PBS
*0.5 ml of 16% PFA
*3.1 ml of ddH2O
 

===== Blocking Buffer =====

Blocking buffer is 5% bovine serum albumin (BSA) in PBS. For 10ml mix 10ml PBS with 0.5g of BSA.
 

===== Permeabilization Buffer: =====

Permeabilization buffer is Blocking Buffer + 0.1% Triton X-100. For 10ml mix 10ml blocking buffer with 10ul Triton X-100.

== GSD Imaging Buffers ==
GSD requires specialized buffers to prevent the triplet state (depleted state) of the fluorophores from being oxidized<ref>http://www.leica-microsystems.com/fileadmin/downloads/Leica%20SR%20GSD/Application%20Notes/Leica_SR_GSD-Protocol_Guide_v1.0.pdf</ref>. This can be achieved by providing a reducing agent (buffer 1) to protect the triplet state, depletion of oxygen in the sample (buffer 2), or a combination of the two (buffer 3). A new buffer has recently been published, but we are still assessing its effectiveness (buffers 4 & 5).

 
===== GSD Buffer 1 (MEA): =====
This is the standard buffer that works for most experiments. It is comprised of PBS, pH 7.4, plus 10mM to 100mM MEA (β-Mercaptoethylamine [AKA Cystamine], Sigma-Aldrich #30070). For 10ml of 50mM/100mM MEA: 

*75mg/150mg of MEA to 10ml of PBS
*MEA will cause the pH of the PBS to change. Return pH to 7.4
*Filter with a 0.22um filter to remove particulates
*Aliquote 1ml into 1.5ml tubes, freeze at -20C 

Use a fresh aliquot each day, do not re-freeze buffer. Frozen aliquots are only good for 1-2 months, after which the buffer must be replaced. Lower MEA concentrations will last for shorter periods of time than higher concentrations.

 

===== GSD Buffer 2 (Glucose-Oxidase): =====
This binary buffer scavenges oxygen from the media. It may work better for some fluorescent proteins than buffer 1, but tends to have higher background fluorescence. 

''Prepare:''

*10x enzyme mix: 5 mg/mL glucose oxidase + 400 μg/mL catalase in PBS + 15% glycerol, pH 7.4. This can be aliquoted and frozen at -20C until needed.
*11% glucose in PBS, pH 7.4. 

''Mounting:''

*For each coverslip, immediately before mounting, pre-mix 10ul of enzyme mix with 90ul of glucose/PBS.
*Mount coverslips as quickly as possible.

 

===== GSD Buffer 3 (Combination): =====
This is a combination of buffers 1 & 2. While used in the literature<ref>Baddeley, D., Crossman, D., Rossberger, S., Cheyne, J. E., Montgomery, J. M., Jayasinghe, I. D., Cremer, C., et al. (2011). 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PloS one, 6(5), e20645. doi:10.1371/journal.pone.0020645</ref>, it is not clear if this offers any additional advantages over either buffer 1 or buffer 2 alone. This buffer may work better for tissue sections. 

''Preparation:''

*Prepare buffer 2 as per usual, but to the glucose/PBS mix add 50mM MEA, and re-pH the solution to 7.4.
*Buffer is mixed and mounted as per buffer 2.

 

===== GSD Buffer 4 (Sulfite Buffer): =====
This is a newer buffer formulation designed for dSTORM<ref>A stable, high refractive index, switching buffer for super-resolution imaging. Tobias M.P. Hartwich, Kenny Kwok Hin Chung, Lena Schroeder, Joerg Bewersdorf, Christian Soeller, David Baddeley. [https://www.biorxiv.org/content/10.1101/465492v1.full bioRxiv 465492; doi: https://doi.org/10.1101/465492]</ref> that has a longer shelf-life and superior imaging characteristics to buffer 1. It requires optimization for specific fluorophores in GSDM and usually does not work "out of the box".

* 10 mM MEA (1.52 mg/mL, 15.2 mg per 10 mL)
* 20 mM Sodium sulfite (2.52 mg/mL, 25.2 mg per 10 mL)
* PBS

Dissolve MEA and Sodium sulfite into PBS. Adjust pH back to pH 7.4 and aliquote. Freeze until needed, should last 2-3 months frozen. Do not reuse aliquots.

 

===== GSD Buffer 4 (AlexaFluor Optimized Buffer): =====
This is GSD Buffer 4 optimized to work well with Alexa Fluor 488, 555, and 647. It also works well with Cy3. This is the best buffer for these fluorophores that we have tested to-date.

* 25 mM MEA (3.75 mg/mL, 37.5 mg per 10 mL)
* 20 mM Sodium sulfite (2.52 mg/mL, 25.2 mg per 10 mL)
* PBS

Dissolve MEA and Sodium sulfite into PBS. Adjust pH back to pH 7.4 and aliquote. Freeze until needed, should last 2-3 months frozen. Do not reuse aliquots.

 
 

== References: ==

<references />

Staining for GSD

2024-08-07T12:32:06Z

Admin: /* GSD Imaging Buffers */

Ground State Depletion (GSD) microscopy detects fluorophore positions independently of intensity or staining density.  As such, it is critical that proper blocking, fixing and staining methods be used in order to avoid off-target fluorescence.  Moreover, coverslips must be of #1.5 (0.17mm) thickness purchased from vendors who can provide coverslips with no fluorescent particulates.  That, or coverslips must be rigorously cleaned (e.g. [http://en.wikipedia.org/wiki/Piranha_solution Piranha cleaning]).  At this time the best coverslips appear to be [http://www.emsdiasum.com/microscopy/products/histology/coverglasses.aspx EMS #72222-01].  As of this writing, coverslips from VWR and Fisher are not GSD-compatible. 

 

Note: careful selection of secondary antibody fluorophores is key to success in GSD.  Make sure you are using established GSD-compatible fluorophores.

== Protocol - Fixation ==

Careful fixation is required to stabalize cell structures while preserving antigenicity and flourescent protein activity.  The following fixation protocol generally works well: 

#Fix cells in 4% PFA in PBS.
#*If cytoskeletal structure is critical, fix for 10min at 10-12C, followed by 20 min at room temperature
#*otherwise, fix for 20 min at room temperature 
#Wash 3X PBS, 30 sec/wash.
#Continue staining as follows:
#*If cells were transfected with fluorescent proteins and no other staining is required, store cells in blocking buffer until imaging. 
#*If intracellular structures are to be immunostained, follow the instructions for intracellular staining.  Use this protocol for both intracellular and mixed intracellular/extracellular staining. 
#*If only extracellular structures are to be immunostained, follow the instructions for extracellular staining. 

== Protocol - Intracellular Staining ==

Use this protocol when immunostaining intracellular structures.  Use this protocol if simultaniously staining intracellular and extracellular structures. 

#Permeabilize and block using permibilization buffer, 1hr at RT.
#*During permeabilization, prepare primary and secondary antibodies in permibilization buffer.  Antibioy concentrations are generally the same as those used for conventional immunoflourescent microscopy. 
#Stain with primary antibody, room temperature, for 1 to 1.5 hours:
#*Use hanging drop method:
#**Put 100ul of antibody(s) in permeabilization buffer on a piece of parafilm.  A highly elevated drop should form. 
#**"Float" coverslip, cell-side-down, on drop. 
#**Cover with foil or box and leave undisturbed (KEEP LEVEL).
#Wash 3X 15min with blocking buffer.
#Add secondary antibody(s) in permeabilization buffer.  Conventional staining or the hanging drop method can be used
#*Secondary staining should be for 1-2 hours, room temperature. 
#Wash 3X 15 min in PBS (no BSA). 
#Post-fix with 2% PFA in PBS, 20 min at room temperature 
#Wash 3X 30 sec with PBS (no BSA) 

 

Cells should be maintained in blocking buffer until imaging.  For imaging cells need to be either mounted in a depression slide using a GSD-appropriate media (e.g. PBS + 50mM cystamine), or coated in PVA. 

== Protocol - Extracellular Staining ==

This protocol should be used only for staining extracellular structures.  If staining both extracellular and intracellular structures, pool the antibodies and follow the intracellular staining protocol, above.

#Block the sample for 1 to 1.5 hours with 5% BSA in PBS
#Stain with primary antibody, room temperature, for 1 to 1.5 hours:
#*Use hanging drop method:
#**Put 100ul of antibody(s) in permeabilization buffer on a piece of parafilm.  A highly elevated drop should form. 
#**"Float" coverslip, cell-side-down, on drop. 
#**Cover with foil or box and leave undisturbed (KEEP LEVEL).
#Wash 3X 15min with PBS + 5% BSA.
#Add secondary antibody(s) in permeabilization buffer.  Conventional staining or the hanging drop method can be used
#*Secondary staining should be for 1-2 hours, room temperature. 
#Wash 3X 15 min in PBS (no BSA). 
#Post-fix with 2% PFA in PBS, 20 min at room temperature 
#Wash 3X 30 sec with PBS + 5% BSA. 

 

== Fluorophore & Fluorescent Protein Selection ==

{| width="80%" cellspacing="1" cellpadding="1" border="1"
|-
| '''Fluorophore''' 
| '''Excitation '''
| '''Emission''' 
| '''Notes '''
|-
| Cy3 
| 550 
| 570 
| Slow to deplete, but blinks forever.  Orange/Red emission. 
|-
| Dylight 649 
| 650 
| 670 
| Best tested fluorophore to-date.  Far-Red emission. 
|-
| YFP 
| 514 
| 527 
| Only confirmed GSD-compatible fluorescent protein at this time.  Cannot be combined with orange/red emitting fluorophores 
|-
| 
| 
| 
| 
|}

 

== Sample Preparation Buffers ==

===== 4% PFA =====

*400 ul of 10x PBS 
*1 ml of 16% PFA 
*2.6 ml of ddH2O
 

===== 2% PFA: =====

1ml of fixative is required for each well in a 12-well plate, or 1.5ml for well in a 12-well plate. For 4ml of PFA mix:

*400 ul of 10x PBS
*0.5 ml of 16% PFA
*3.1 ml of ddH2O
 

===== Blocking Buffer =====

Blocking buffer is 5% bovine serum albumin (BSA) in PBS.  For 10ml mix 10ml PBS with 0.5g of BSA.
 

===== Permeabilization Buffer: =====

Permeabilization buffer is Blocking Buffer + 0.1% Triton X-100. For 10ml mix 10ml blocking buffer with 10ul Triton X-100.

== GSD Imaging Buffers ==
GSD requires specialized buffers to prevent the triplet state (depleted state) of the fluorophores from being oxidized<ref>http://www.leica-microsystems.com/fileadmin/downloads/Leica%20SR%20GSD/Application%20Notes/Leica_SR_GSD-Protocol_Guide_v1.0.pdf</ref>. This can be achieved by providing a reducing agent (buffer 1) to protect the triplet state, depletion of oxygen in the sample (buffer 2), or a combination of the two (buffer 3). A new buffer has recently been published, but we are still assessing its effectiveness (buffers 4 & 5).

 
===== GSD Buffer 1 (MEA): =====
This is the standard buffer that works for most experiments. It is comprised of PBS, pH 7.4, plus 10mM to 100mM MEA (β-Mercaptoethylamine [AKA Cystamine], Sigma-Aldrich #30070). For 10ml of 50mM/100mM MEA: 

*75mg/150mg of MEA to 10ml of PBS 
*MEA will cause the pH of the PBS to change. Return pH to 7.4 
*Filter with a 0.22um filter to remove particulates 
*Aliquote 1ml into 1.5ml tubes, freeze at -20C 

Use a fresh alloquot each day, do not re-freeze buffer.  Frozen aliquotes are only good for 1-2 months, after which the buffer must be replaced.  Lower MEA concentrations will last for shorter periods of time than higher concentrations.

 

===== GSD Buffer 2 (Glucose-Oxidase): =====
This binary buffer scavenges oxygen from the media.  It may work better for some fluorescent proteins than buffer 1, but tends to have higher background fluorescence. 

''Prepare:'' 

*10x enzyme mix: 5 mg/mL glucose oxidase + 400 μg/mL catalase in PBS + 15% glycerol, pH 7.4.  This can be aliquoted and frozen at -20C until needed. 
*11% glucose in PBS, pH 7.4. 

''Mounting:'' 

*For each coverslip, immediately before mounting, pre-mix 10ul of enzyme mix with 90ul of glucose/PBS. 
*Mount coverslips as quickly as possible.

 

===== GSD Buffer 3 (Combination): =====
This is a combination of buffers 1 & 2. While used in the literature<ref>Baddeley, D., Crossman, D., Rossberger, S., Cheyne, J. E., Montgomery, J. M., Jayasinghe, I. D., Cremer, C., et al. (2011). 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PloS one, 6(5), e20645. doi:10.1371/journal.pone.0020645</ref>, it is not clear if this offers any additional advantages over either buffer 1 or buffer 2 alone. This buffer may work better for tissue sections. 

''Preparation:''

*Prepare buffer 2 as per usual, but to the glucose/PBS mix add 50mM MEA, and re-pH the solution to 7.4.
*Buffer is mixed and mounted as per buffer 2.

 

===== GSD Buffer 4 (Sulfite Buffer): =====
This is a newer buffer formulation designed for dSTORM<ref>A stable, high refractive index, switching buffer for super-resolution imaging. Tobias M.P. Hartwich, Kenny Kwok Hin Chung, Lena Schroeder, Joerg Bewersdorf, Christian Soeller, David Baddeley. [https://www.biorxiv.org/content/10.1101/465492v1.full bioRxiv 465492; doi: https://doi.org/10.1101/465492]</ref> that has a longer shelf-life and superior imaging characteristics to buffer 1. It requires optimization for specific fluorophores in GSDM and usually does not work "out of the box".

* 10 mM MEA (1.52 mg/mL, 15.2 mg per 10 mL)
* 20 mM Sodium sulfite (2.52 mg/mL, 25.2 mg per 10 mL)
* PBS

Dissolve MEA and Sodium sulfite into PBS. Adjust pH back to pH 7.4 and aliquote. Freeze until needed, should last 2-3 months frozen. Do not reuse aliquots.

 

===== GSD Buffer 4 (AlexaFluor Optimized Buffer): =====
This is GSD Buffer 4 optimized to work well with Alexa Fluor 488, 555, and 647. It also works well with Cy3. This is the best buffer for these fluorophores that we have tested to-date.

* 25 mM MEA (1.52 mg/mL, 15.2 mg per 10 mL)
* 20 mM Sodium sulfite (2.52 mg/mL, 25.2 mg per 10 mL)
* PBS

Dissolve MEA and Sodium sulfite into PBS. Adjust pH back to pH 7.4 and aliquote. Freeze until needed, should last 2-3 months frozen. Do not reuse aliquots.

 
 

== References: ==

<references />

Staining for GSD

2024-08-07T12:31:06Z

Admin: /* Recipes */

Ground State Depletion (GSD) microscopy detects fluorophore positions independently of intensity or staining density.  As such, it is critical that proper blocking, fixing and staining methods be used in order to avoid off-target fluorescence.  Moreover, coverslips must be of #1.5 (0.17mm) thickness purchased from vendors who can provide coverslips with no fluorescent particulates.  That, or coverslips must be rigorously cleaned (e.g. [http://en.wikipedia.org/wiki/Piranha_solution Piranha cleaning]).  At this time the best coverslips appear to be [http://www.emsdiasum.com/microscopy/products/histology/coverglasses.aspx EMS #72222-01].  As of this writing, coverslips from VWR and Fisher are not GSD-compatible. 

 

Note: careful selection of secondary antibody fluorophores is key to success in GSD.  Make sure you are using established GSD-compatible fluorophores.

== Protocol - Fixation ==

Careful fixation is required to stabalize cell structures while preserving antigenicity and flourescent protein activity.  The following fixation protocol generally works well: 

#Fix cells in 4% PFA in PBS.
#*If cytoskeletal structure is critical, fix for 10min at 10-12C, followed by 20 min at room temperature
#*otherwise, fix for 20 min at room temperature 
#Wash 3X PBS, 30 sec/wash.
#Continue staining as follows:
#*If cells were transfected with fluorescent proteins and no other staining is required, store cells in blocking buffer until imaging. 
#*If intracellular structures are to be immunostained, follow the instructions for intracellular staining.  Use this protocol for both intracellular and mixed intracellular/extracellular staining. 
#*If only extracellular structures are to be immunostained, follow the instructions for extracellular staining. 

== Protocol - Intracellular Staining ==

Use this protocol when immunostaining intracellular structures.  Use this protocol if simultaniously staining intracellular and extracellular structures. 

#Permeabilize and block using permibilization buffer, 1hr at RT.
#*During permeabilization, prepare primary and secondary antibodies in permibilization buffer.  Antibioy concentrations are generally the same as those used for conventional immunoflourescent microscopy. 
#Stain with primary antibody, room temperature, for 1 to 1.5 hours:
#*Use hanging drop method:
#**Put 100ul of antibody(s) in permeabilization buffer on a piece of parafilm.  A highly elevated drop should form. 
#**"Float" coverslip, cell-side-down, on drop. 
#**Cover with foil or box and leave undisturbed (KEEP LEVEL).
#Wash 3X 15min with blocking buffer.
#Add secondary antibody(s) in permeabilization buffer.  Conventional staining or the hanging drop method can be used
#*Secondary staining should be for 1-2 hours, room temperature. 
#Wash 3X 15 min in PBS (no BSA). 
#Post-fix with 2% PFA in PBS, 20 min at room temperature 
#Wash 3X 30 sec with PBS (no BSA) 

 

Cells should be maintained in blocking buffer until imaging.  For imaging cells need to be either mounted in a depression slide using a GSD-appropriate media (e.g. PBS + 50mM cystamine), or coated in PVA. 

== Protocol - Extracellular Staining ==

This protocol should be used only for staining extracellular structures.  If staining both extracellular and intracellular structures, pool the antibodies and follow the intracellular staining protocol, above.

#Block the sample for 1 to 1.5 hours with 5% BSA in PBS
#Stain with primary antibody, room temperature, for 1 to 1.5 hours:
#*Use hanging drop method:
#**Put 100ul of antibody(s) in permeabilization buffer on a piece of parafilm.  A highly elevated drop should form. 
#**"Float" coverslip, cell-side-down, on drop. 
#**Cover with foil or box and leave undisturbed (KEEP LEVEL).
#Wash 3X 15min with PBS + 5% BSA.
#Add secondary antibody(s) in permeabilization buffer.  Conventional staining or the hanging drop method can be used
#*Secondary staining should be for 1-2 hours, room temperature. 
#Wash 3X 15 min in PBS (no BSA). 
#Post-fix with 2% PFA in PBS, 20 min at room temperature 
#Wash 3X 30 sec with PBS + 5% BSA. 

 

== Fluorophore & Fluorescent Protein Selection ==

{| width="80%" cellspacing="1" cellpadding="1" border="1"
|-
| '''Fluorophore''' 
| '''Excitation '''
| '''Emission''' 
| '''Notes '''
|-
| Cy3 
| 550 
| 570 
| Slow to deplete, but blinks forever.  Orange/Red emission. 
|-
| Dylight 649 
| 650 
| 670 
| Best tested fluorophore to-date.  Far-Red emission. 
|-
| YFP 
| 514 
| 527 
| Only confirmed GSD-compatible fluorescent protein at this time.  Cannot be combined with orange/red emitting fluorophores 
|-
| 
| 
| 
| 
|}

 

== Sample Preparation Buffers ==

===== 4% PFA =====

*400 ul of 10x PBS 
*1 ml of 16% PFA 
*2.6 ml of ddH2O
 

===== 2% PFA: =====

1ml of fixative is required for each well in a 12-well plate, or 1.5ml for well in a 12-well plate. For 4ml of PFA mix:

*400 ul of 10x PBS
*0.5 ml of 16% PFA
*3.1 ml of ddH2O
 

===== Blocking Buffer =====

Blocking buffer is 5% bovine serum albumin (BSA) in PBS.  For 10ml mix 10ml PBS with 0.5g of BSA.
 

===== Permeabilization Buffer: =====

Permeabilization buffer is Blocking Buffer + 0.1% Triton X-100. For 10ml mix 10ml blocking buffer with 10ul Triton X-100.

== GSD Imaging Buffers ==
GSD requires specialized buffers to prevent the triplet state (depleted state) of the fluorophores from being oxidized<ref>http://www.leica-microsystems.com/fileadmin/downloads/Leica%20SR%20GSD/Application%20Notes/Leica_SR_GSD-Protocol_Guide_v1.0.pdf</ref>. This can be achieved by providing a reducing agent (buffer 1) to protect the triplet state, depletion of oxygen in the sample (buffer 2), or a combination of the two (buffer 3). A new buffer has recently been published, but we are still assessing its effectiveness (buffers 4 & 5).
 
===== GSD Buffer 1 (MEA): =====
This is the standard buffer that works for most experiments. It is comprised of PBS, pH 7.4, plus 10mM to 100mM MEA (β-Mercaptoethylamine [AKA Cystamine], Sigma-Aldrich #30070). For 10ml of 50mM/100mM MEA: 

*75mg/150mg of MEA to 10ml of PBS 
*MEA will cause the pH of the PBS to change. Return pH to 7.4 
*Filter with a 0.22um filter to remove particulates 
*Aliquote 1ml into 1.5ml tubes, freeze at -20C 

Use a fresh alloquot each day, do not re-freeze buffer.  Frozen aliquotes are only good for 1-2 months, after which the buffer must be replaced.  Lower MEA concentrations will last for shorter periods of time than higher concentrations. 

===== GSD Buffer 2 (Glucose-Oxidase): =====
This binary buffer scavenges oxygen from the media.  It may work better for some fluorescent proteins than buffer 1, but tends to have higher background fluorescence. 

''Prepare:'' 

*10x enzyme mix: 5 mg/mL glucose oxidase + 400 μg/mL catalase in PBS + 15% glycerol, pH 7.4.  This can be aliquoted and frozen at -20C until needed. 
*11% glucose in PBS, pH 7.4. 

''Mounting:'' 

*For each coverslip, immediately before mounting, pre-mix 10ul of enzyme mix with 90ul of glucose/PBS. 
*Mount coverslips as quickly as possible.

 

===== GSD Buffer 3 (Combination): =====
This is a combination of buffers 1 & 2. While used in the literature<ref>Baddeley, D., Crossman, D., Rossberger, S., Cheyne, J. E., Montgomery, J. M., Jayasinghe, I. D., Cremer, C., et al. (2011). 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PloS one, 6(5), e20645. doi:10.1371/journal.pone.0020645</ref>, it is not clear if this offers any additional advantages over either buffer 1 or buffer 2 alone. This buffer may work better for tissue sections. 

''Preparation:''

*Prepare buffer 2 as per usual, but to the glucose/PBS mix add 50mM MEA, and re-pH the solution to 7.4.
*Buffer is mixed and mounted as per buffer 2.

 

===== GSD Buffer 4 (Sulfite Buffer): =====
This is a newer buffer formulation designed for dSTORM<ref>A stable, high refractive index, switching buffer for super-resolution imaging. Tobias M.P. Hartwich, Kenny Kwok Hin Chung, Lena Schroeder, Joerg Bewersdorf, Christian Soeller, David Baddeley. [https://www.biorxiv.org/content/10.1101/465492v1.full bioRxiv 465492; doi: https://doi.org/10.1101/465492]</ref> that has a longer shelf-life and superior imaging characteristics to buffer 1. It requires optimization for specific fluorophores in GSDM and usually does not work "out of the box".

* 10 mM MEA (1.52 mg/mL, 15.2 mg per 10 mL)
* 20 mM Sodium sulfite (2.52 mg/mL, 25.2 mg per 10 mL)
* PBS

Dissolve MEA and Sodium sulfite into PBS. Adjust pH back to pH 7.4 and aliquote. Freeze until needed, should last 2-3 months frozen. Do not reuse aliquots.

 

===== GSD Buffer 4 (AlexaFluor Optimized Buffer): =====
This is GSD Buffer 4 optimized to work well with Alexa Fluor 488, 555, and 647. It also works well with Cy3. This is the best buffer for these fluorophores that we have tested to-date.

* 25 mM MEA (1.52 mg/mL, 15.2 mg per 10 mL)
* 20 mM Sodium sulfite (2.52 mg/mL, 25.2 mg per 10 mL)
* PBS

Dissolve MEA and Sodium sulfite into PBS. Adjust pH back to pH 7.4 and aliquote. Freeze until needed, should last 2-3 months frozen. Do not reuse aliquots.
 

== References: ==

<references />

THP1 Culture and Differentiation

2024-07-29T19:04:56Z

Admin:

THP1 Culture and Differentiation

2024-07-29T19:04:00Z

Admin:

2022-03-02T18:42:43Z

Admin:

2022-03-02T17:37:11Z

Admin:

File:18mm Lid Clear.png

2022-03-02T17:36:38Z

Admin:

18mm Lid Clear

File:18mm Lid Solid.png

2022-03-02T17:36:25Z

Admin:

18mm Align

File:18mm Align.png

2022-03-02T17:36:08Z

Admin:

18mm Align

File:50mm Lid Clear.png

2022-03-02T17:35:48Z

Admin:

50mm Lid Clear

File:50mm Lid Solid.png

2022-03-02T17:35:31Z

Admin:

50mm Lid Solid