Immunofluorescence

Freeze a small quantity of acetone at -20C 

Remove slides from -80C freezer and place into -20C acteone. Keep container with slides in acetone in -20C freezer for 20min incubation.

All subsequent steps at room temperature:
Rinse in PBS 
Rehydrate in PBS for 10min 

Dry edges of the slide with kimwipes and draw circle around tissue with a hydrophobic barrier pen, such as ImmEdge.  
Block for 1hr using 200ul PBS+10% rat serum. 

DIlute primary antibody 1:100 in PBS + 1% BSA
Tap blocking buffer off the slide onto paper towel. 

Add 150ul primary Ab and incubate for 1hr in the dark. 
Rinse in PBS for 5min. Repeat 2 more times with fresh PBS.

Dilute secondary antibody 1:1000 in PBS + 1% BSA.

Add 150ul secondary Ab and incubate for 1hr in the dark. 

Rinse in PBS for 5min. Repeat 2 more times with fresh PBS.
Dilute DAPI 1:10000 (from 1mg/ml stock) into PBS. Add 200ul and incubate for 5 min.
Rinse slides with PBS.
Rinse in PBS for 5 mins.
Remove excess PBS and add mounting media to slides and add coverslips