Solutions to prepare:

TBS 1X: Alternative to PBS, but necessary as c-Fos is a Phospho-protein. Prepare from 10X solution. Example: 1L of TBS X1 = 100ml of TBS X10 + 900ml dH20

TBST: TBS X1 + 0.2% of Triton X100. Mix well. Example: 100ml of TBST = 200l Triton X100 + 100ml of TBS X1

TBST + NDS: 5% v/v Normal Donkey Serum + TBST. Mix well. Example: 25ml of TBST + NDS = 1.25ml NDS + 23.75ml TBST NOTE: NDS is rehydrated with dH20 and then 1:20 dilution made from rehydrated solution

DAY 1:

Step 1: Gather desired slides into a 4×6 well plate. Cut a transfer pipette to allow wider width and use this to collect slides and anti-freeze from storage well plates to working well plate. Have two wells designated as controls, one without primary antibody, one without secondary antibody.

Step 2: Wash slides in TBST for 10 min, 3 times on agitator (room temp.). Remove the liquid from each well with a thinned transfer pipette and then fill the wells around half way (~1ml) with TBST.

Step 3: Block in TBST + NDS (1ml/well) for 60 min on agitator in fridge.

Step 4: While waiting for block, prepare primary antibody. c-Fos antibody 1/8000 concentration in TBST + NDS (1/8000 = 0.125μl of primary in 1ml of TBST + NDS. Example: 36 wells = 0.125μl * 36 = 4.5μl of primary + (36000μl – 4.5μl) 35995.5μl of TBST + NDS = TOTAL volume of 36000μl (36ml).

Step 5: Remove block and then add in solution containing primary antibody (1ml/well) to all wells except for primary control (add in TBST + NDS to control well). Leave in fridge on very slow agitation overnight (not 24 h later).

DAY 2:

Step 1: Wash slides in TBST for 10 min, 3 times on agitator (room temp.). Remove the liquid from each well with a thinned transfer pipette and then fill the wells around half way (~1ml) with TBST.

Step 2: Prepare secondary antibody. Donkey anti-Rabbit, Alexa 488 1/200 concentration in TBST (1/200 = 5ul of secondary in 1ml of TBST) Example: 36 wells = 5μl * 36 = 180μl of primary + (36000μl – 180μl) 35820μl of TBST = TOTAL volume of 36000μl (36ml). NOTE: Alexa 488 is light-sensitive, prepare solutions in dark and hold in a falcon tube covered in aluminium foil.

Step 3: Remove TBST and introduce solution secondary antibody (1ml/well) to all wells except for secondary control (add in TBST to this control well). Cover plate in foil and put on slow agitation in the fridge for 1 h.

Step 4: In a dark room wash slides in TBS for 10 min, 3 times on agitator (room temp.). Remove the liquid from each well with a thinned transfer pipette and then fill the wells around half way (~1ml) with TBS.

Step 5: Fill a flat circular dish with TBS and transfer the desired slides (one well at a time) into the container. From there, mount slides onto a gelled microscope slide. Add Fluromount-G+DAPI (kept in fridge – about 1 drop per tissue section) and then place a coverslip on top. Allow slide to dry in a dark place. When ready, place slides in slide holder and store in fridge until imaging.