1. Take up bulk quantities of diaminobenzidine tetrahydrochloride dehydrate in water and bulk quantities of the free base in 0.1 M hydrochloric acid so that the concentration of DAB does not exceed 0.9 mg/ml. Dilute solutions with the same buffer, if necessary, so that the concentration does not exceed 0.9 mg/ml.
2. For each 10 ml of solution, add 5 ml of 0.2 M potassium permanganate solution (31.6 g KMnO4 per liter of solution with water) and 5 ml of 2 M sulfuric acid solution (112 ml concentrated H2S04 per liter of solution with water).
3. Allow the mixture to stand for at least 10 hours.
4. Test to ensure pH of solution is between 6-9. Dispose solution down the drain with copious amounts of water.

In molecular biology, c-Fos is a cellular proto-oncogene belonging to the immediate early gene family of transcription factors. c-Fos has a leucine-zipper DNA binding domain, and a transactivation domain at the C-terminus. Transcription of c-Fos is upregulated in response to many extracellular signals, e.g. growth factors. Additionally, phosphorylation by MAPK, PKA, PKC or cdc2 alters the activity and stability of c-Fos. Members of the Fos family dimerise with Jun to form the AP-1 transcription factor, which upregulates transcription of a diverse range of genes involved in everything from proliferation and differentiation to defense against invasion and cell damage.

The AP-1 complex has been implicated in transformation and progression of cancer, and both Fos and Jun were first discovered in rat fibroblasts.

The viral homologue of c-Fos, v-Fos, is found in the retrovirus Finkel-Biskis-Jinkins murine osteogenic sarcoma virus. In neuroscience research, neuroscientists measure expression of c-fos as an indirect marker of neuronal activity because c-fos is often expressed when neurons fire action potentials.

Staining procedure

1. This is a free-floating staining procedure for formalin-fixed brain tissue. Sections should be cut between 15-30 µm.
2. Transfer sections in 6-well plates loaded with PBS 0.1 M (one brain per well).
3. Rinse sections twice, 10 minutes each rinse, with PBS 0.1 M on a shaker.
4. Incubate sections with fresh 0.3% H₂O₂ in PBS 0.1 M for 30 minutes at room temperature on a shaker.
5. Rinse sections 3 x 10 minutes with PBS 0.1 M on a shaker.
6. Incubate sections with blocking solution  for 60 min at room temperature on a shaker.
7. Incubate sections with primary antibody diluted in blocking solution overnight at room temperature on a shaker.  With certain antibodies, to reduce background staining, consider an incubation for 2-3 days at 4°C.
8. Rinse sections 4 x 10 minutes with PBS 0.1 M on a shaker.
9. Incubate sections with biotinylated secondary antibody, diluted in blocking solution for 2 hours at room temperature on a shaker.
10. Rinse sections 4 x 10 minutes in PBS 0.1 M on a shaker.
11. Prepare ABC solution  at least 30 minutes prior to incubation to allow for ABC complex to form. Add 2 drops of solution A and 2 drops of solution B per 10 ml of blocking solution. Solutions A and B can also be added to plain PBS 0.1 M.
12. Incubate sections in ABC solution for 1-2 hours at room temperature on a shaker.
13. Rinse sections 4 x 10 minutes with PBS 0.1 M on a shaker.
14. Incubate sections in DAB solution for 8 minutes at room temperature on a shaker. DAB solution is highly toxic and carcinogen. Wear gloves and handle with care.
15. Add three drops of 0.3%  H₂O₂ (~125 ul) to each well to reveal staining. When background is satisfactory (after 1 to 5 min), halt the reaction by adding PBS 0.1 M.
16. Rinse sections 4 x 10 minutes with PBS 0.1 M on a shaker.
17. Transfer sections to slides using a brush, allow to air dry. It is best to transfer sections as soon as possible but well plates can be stored for a few days in the fridge at 4°C.
18. Dehydrate slides twice in ethanol 100% for 5 minutes each.
19. Incubate slides twice in toluene or xylene for 5 minutes each.
20. Add mounting medium to slides while still wet. Place coverslips to slides and allow to dry. Examine staining by microscopy.

Reagents

• Sodium phosphate, monobasic anhydrous NaH₂PO₄ (FW 120.0). Sigma,  S-0751, 1Kg
• Sodium phosphate, dibasic anhydrous, Na₂HPO₄ (FW 142.0). Sigma, S-0876, 1Kg
• Hydrogen peroxide, H2O2  30% (w/w) solution. Sigma, H-1009, 100 ml
• Albumin Bovine fraction V, min 96%, electrophoresis. Sigma, A-9647, 50g
• 3,3′-diaminobenzidine tablets (DAB). Sigma, D-5905, 50 tablets
• Goat serum. BioWest, Cat# S2000, 100ml or similar
• Vectastain ABC Kit, Elite standard. Vector, PK-6100
• Triton X-100 (t-Octylphenoxypolyethoxyethanol). Sigma, T-9284, 100 ml
• Toluene or xylene from VWR or Fisher
• Ethanol 100%

Antibodies

Titrate new batches of antibodies for appropriate concentration before using in experiments as effective concentrations may vary across batches of antibody?.

Primary

Rabbit anti-Fos polyclonal IgG, Oncogene Research Products (Ab-5, Cat.# PC38). Recommended dilution, 1:20 000.

Secondary

Biotin-SP-conjugated affiniPure Goat anti-rabbit IgG (H+L) (minimal cross reaction to Human , Mouse and rat serum proteins). Made in goat. Jackson Immunoresearch, Cat.# 111-065-144. Recommended dilution: 1:2000.

Solutions

Phosphate buffer solution, 0.2 M,  pH 7.4

1. Collect 1000 ml of distilled water in a graduated cylinder. Pour about 400 ml of water in a beaker and stir.
2. Weigh 4.8 g of Sodium Phosphate monobasic NaH₂PO₄ and 22.72 g of sodium phosphate dibasic Na₂HPO₄ .
3. Add to the 400 ml of water. When dissolved, add the rest of the water and continue stirring for 5 min. Take pH which should be around 7.4.

Phosphate buffer solution, 0.1 M,  pH 7.4

Make phosphate buffer 0.2M solution as described above and add 1000 ml of distilled water to bring it to 0.1 M, total volume 2 liters. pH should be around 7.4. Solution can be kept at room temperature or at 4°C.

Blocking solution (PBS 0.1 M; 0.1 % BSA; 0.2% Triton X-100; 2% serum)

Collect about 800 ml of phosphate buffer 0.1 M in a graduated cylinder. Add 20 ml of serum, 2 ml of Triton X-100 and 1 g of BSA. Stir for 10 min. Add more PBS 0.1 M to reach 1000 ml. Stir another 5 min. Store blocking solution in 50-ml aliquots (50-ml Falcon tubes) at -20°C

DAB solution, 0.05% (w/v)

Add 1 tablet (10 mg) of DAB in 20 ml of PBS 0.1 M in a 50-ml Falcon tube. Vortex vigorously until dissolved. Solution should be used fresh, or may be frozen in single-use aliquots and stored at -20C until use. Wear gloves and inactivate solution using a 10% bleach solution (dilute DAB with an equal volume of bleach) when finished and dispose in appropriate biohazard container. DAB is highly toxic and carcinogen; do not dump solution down the drain without treatment.

Neutralization of DAB

Although chlorine bleach is commonly employed in many laboratories as a neutralization procedure, it is not effective in removing the mutagenic properties of DAB. A potassium permanganate-sulfuric acid procedure must be used.

1. Take up bulk quantities of diaminobenzidine tetrahydrochloride dehydrate in water and bulk quantities of the free base in 0.1 M hydrochloric acid so that the concentration of DAB does not exceed 0.9 mg/ml.  Dilute solutions with the same buffer, if necessary, so that the concentration does not exceed 0.9 mg/ml.
2. For each 10 ml of solution, add 5 ml of 0.2 M potassium permanganate solution and 5 ml of 2 M sulfuric acid solution.
3. Allow the mixture to stand overnight, decolorize by the addition of sodium ascorbate, neutralize and dispose solution down the drain with copious amounts of water.

0.3% (v/v) H₂O₂ solution

Add 0.5 ml of H₂O₂ 30% solution to 50 ml of PBS 0.1 M in a 50-ml Falcon tube. Vortex. Use fresh.

Equipment

• Microscope
• 2D Shaker
• 6-well plates
• Gelatin-coated slides or precleaned superfrost plus slides (25 x 75 x 1 mm). VWR, Cat.# 48311-703
• Coverlips (micro cover glasses) 24 x 60 mm, No. 1. VWR, Cat.# 48404 454.
• Mounting medium (Eukit or Cytoseal 280 from Richard-Allan Scientific (8311-4) or similar)