Spitzer ICC Protocol Template

Day1 ICC Date 

Cells plated by on date 

Step 1: Make fresh 4% PFA  filter 

Step 2: Wash cells 2x PBS(TC) (500ul/well) 12

Step 3: Fix PFA 15 min 500ul/well 

Step 4: Wash 2x PBS(ICC from here out) 12

Step 5: PBTx 10 min (500ul/well) 

Step 6: Wash 3x PBS, 12move to humidity chamber before last wash 3

Step 7: Block 100ul per coverslip >1hr start  end 

Block Solution: 5% BSA (0.25g/5mL) in 10% GS

*****enter math here********         

Step 8: Primary in Block 100ul per coverslip 1:500 ON fridge

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Day 2 Date

Step 1: Wash 6x 5min PBS 123456

Step 2: BSA solution for secondary Ab: 5%BSA in 1%GS in PBS

******enter math here ******

Step 3: Secondary solution: 100ul per coverslip Antibodies at 1:500, phalloidin at 1:40 ON fridge

****** enter specifics here ******

Day 3 Date

Step 1: Take Prolong Gold out of freezer to warm up; keep in dark 

Step 2: Wash 5x 5min PBS 12345

Step 3: DAPI in PBS at 5:1000, 100ul per coverslip, 5min 

Step 4: One at at time, dip each coverslip in mqH2O, mount with 8ul Prolong Gold on clean slide 

Step 5: Allow to cure flat in dark ON 

Step 6: Store in slide box in fridge