Immunocytochemistry (ICC)

Neurons - ICC

Day 1

IF Final [Chapman Lab]

• 4% PFA - (9ml+9ml=16%;4+12ml=4%)10mL)0.4g PFA in 10mL 1x PBS, heat at 60C for ~30min, invert occasionally, pH 7.3, filter sterilize, freeze
• Perm/quench - (10mL) 9.7 mL 1x PBS + 100 uL 5M NH4Cl(50mM final), 200 uL 10% Saponin in PBS (0.2% final)
• Block 1 10% BSA - (10mL) 40uL of 10% Saponin(.04% final), 10 uL of 20% azide (.02% final)+ 1g BSA (10% final) + 1ml of Goat serum (secondary matchedserum block) + 8.95 mL 1x PBS
• Block 2 1% BSA - (10mL) 40uL of 10% Saponin(.04% final), 10 uL of 20% azide(.02% final) + 0.1g BSA (1% final) + 9.95 mL 1x PBS
• Wash - [10mL] 40uL of 10% Saponin (.04% final), 10 uL of 20% azide (.02% final) + 9.95 mL 1x PBS

1. Prewarm fix in incubator (37C/5% CO2)
2. Fix 1O min in incubator (remainder at RT) 15:00 
   1. wash 1x quick
   2. 1x 5min w/ PBS 05:00 
3. Perm/quench 10 min 10:00
   1. wash 1x quick 
   2. 1x 5min w/ PBS 05:00 
4. Block 1 hr (block 1) 01:00:00
5. Primary 1h or o/n @ 4C (depending on antibody), diluted in block 2 01:00:00
   1. wash 1x quick,
   2. wash 1x 5min 05:00
   3. wash 1x 5min 05:00
   4. wash 1x 5min 05:00
6. Secondary 1 hr, diluted in block 2 01:00:00
   1. wash 1x quick,
   2. 1x 5min w/ PBS 05:00
   3. 1x 5min w/PBS 05:00
   4. 1x 5min w/PBS 05:00
7. Mount o/n

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