https://wiki.phagocytes.ca/index.php?action=history&feed=atom&title=Ablation_of_Recycling_Endosomes 2026-04-28T14:58:44Z Revision history for this page on the wiki MediaWiki 1.40.1 https://wiki.phagocytes.ca/index.php?title=Ablation_of_Recycling_Endosomes&diff=57&oldid=prev 2021-02-01T19:54:20Z

<p>Created page with &quot;From [http://www.ncbi.nlm.nih.gov/pubmed/15534004 Ang et al. 2004, JCB 167: 531] == &lt;br&gt; Stock solutions == ===== Human Tfn-HRP ===== *0.5 mg/ml in H2O + 20% glycerol ===...&quot;</p> <p><b>New page</b></p><div>From [http://www.ncbi.nlm.nih.gov/pubmed/15534004 Ang et al. 2004, JCB 167: 531]<br /> <br /> == &lt;br&gt; Stock solutions ==<br /> <br /> ===== Human Tfn-HRP =====<br /> <br /> *0.5 mg/ml in H2O + 20% glycerol <br /> <br /> ===== Acid Wash =====<br /> <br /> *150 mM NaCl <br /> *20 mM citric acid <br /> <br /> pH 5.0, adjust with NaOH<br /> <br /> ===== DAB solution =====<br /> <br /> weigh freshly to make a 10 mg/ml in PBS solution -&amp;gt; Dilute 1:100 to make 0.1 mg/ml DAB in PBS working solution, PROTECT FROM LIGHT<br /> <br /> ===== Other =====<br /> <br /> 30% H2O2&lt;br&gt; 1% BSA in PBS<br /> <br /> == Protocol ==<br /> <br /> ===== Tfn labeling =====<br /> <br /> *Serum starve VSVG-transfected cells in SF-DMEM 1h at 40 ºC <br /> *Tfn-HRP 0.01 mg/ml (1:50 from 0.5 mg/ml stock) 45 min 40 ºC in SF-DMEM <br /> *If fluorescent transferrin to be added: 50 ug/ml Tfn-Alexa (1:100 from 5 mg/ml stock) 45 min 40 ºC in SF-DMEM together with Tfn-HRP <br /> *Wash SF-DMEM <br /> *Chase 20 min 40 ºC <br /> <br /> ===== Ablation on ice =====<br /> <br /> #Wash 2x ice-cold PBS <br /> #Wash 2x 5 min with acid wash to remove surface-bound Tfn-HRP <br /> #Wash 2x ice-cold PBS <br /> #Incubate cells for 1 hr on ice in the dark with rocking as follows: <br /> #*ABLATION: 0.1 mg/ml DAB + 0.025% H2O2 (8.4 µl 30% solution per 10 ml DAB in PBS) <br /> #*CONTROL: 0.1 mg/ml DAB, no H2O2 <br /> #Wash 2x 1% BSA in PBS <br /> <br /> ===== Chase&lt;br&gt; =====<br /> <br /> #Add warm HPMI + 100 ug/ml cycloheximide, chase at 32 ºC 1.5 h <br /> #Wash PBS, FIX <br /> #VSVG trafficks to PM in control cells, in ablated cells remains in Golgi/RE</div>

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