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immunocytochemistry_protocol

immunocytochemistry_protocol - Immunocytochemistry Protocol...

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Immunocytochemistry Protocol Day 2 1. Aspirate off stimulant and add 1ml ice cold 4%paraformaldehyde sol. a. 1g paraformaldehyde {FOR 24- WELLS} b. 1 pellet NaOH c. 18ml H 2 O(once in sol): d. 2.5 ml 10X PBS e. .038g EGTA 4mM f. ph to 7.4 and filter sterilize(bring up to 25ml) 2. Incubate for 30 min at RT 3. Washing the cells a. Aspirate off paraformaldehyde b. Wash w/ 500ul of 1X PBS/100mM glycine 2x for 5 min (glycine should be warmed to RT) i. 10 ml 10X PBS ii. .75g glycine 100mM iii. 90mls H 2 O (store sol @ 4 o ) 4. Blocking the cells a. Make blocking agent i. 1X PBS/2% serum/0.4%saponin 1. 600ul 2% goat serum 2. .12g of .4% saponin (detergent) 3. 29.28 ml 1X PBS (should total 30ml sol) b. Aspirate off PBS/G washing sol c. Add 500ul of blocking agent to each well d. Incubate for 1hr at RT 5. Adding Primary antibody {SERT=1:1000;TROPH=1:100} a. After incubation remove coverslip from well and place on the parafilm humidifier box b. Immediately add 50ul of the primary abs in blocking reagent to each coverslip c. Incubate overnight @4 o
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Immunocytochemistry Protocol Day 2 6. Washing the Cells a. Place coverslips back into a 24-well plate that
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