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 = 200μl 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:

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

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.
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.
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.
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.
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.