Description

Weil’s stain is a modification for paraffin sections of the Weigert-Pal-Kulschitsky technique. The underlying principle of these methods involves the reduction of chrome salt to chromium dioxide by myelin. The chromium subsequently acts as a mordant for the haematoxylin, intensifying the stain.

Procedure

This procedure is generally conducted on sections from formalin-fixed, paraffin-embedded tissue that are cut between 8-15 µm. Spinal cord tissue is rich in myelinated axons and can be used as a positive control.

1. Dewax and hydrate sections to distilled water.
2. Put slides in freshly prepared Staining Solution at 56-60C for 30 minutes.
3. Wash slides well in water.
4. Partially differentiate in iron alum differentiating solution until myelin sheaths stand out blueish-black on a pale grey background, approximately 5 minutes. If you are unsure, check your sections under the microscope at1 minute intervals).
5. Wash slides in tap water for 10 minutes.
6. Complete differentiation in Weigert’s differentiator, 1 to 2 minutes. Control this differentiation step carefully checking under the microscope, until the myelin is an intense deep blue  against a creamy or clear background.
7. Wash well in tap water.
8. Dehydrate through a series of graded ethanol baths, clear in xylene, and mount.

RESULTS

Myelin-containing structures will be stained black, red blood cells will be black, nuclei will be blue, and the background should be clear or yellow.

Solutions

Haematoxylin Solution, working strength

• 10g Haematoxylin
• 100 ml absolute ethanol.

Allow solution to “ripen” naturally for six (6) weeks, forming the basis of a stock solution. Just prior to staining, create a working strength solution by diluting the stock 1:4 with distilled water

Iron Alum Solution

• 4% (w/v) aqueous ferric ammonium sulphate.

Staining Solution

Mix equal volumes of preheated (56-60C) working strength Haematoxylin and Iron Alum solutions just prior to use.

Weigert’s Differentiator, 200 ml

• Borax (sodium tetraborate), 2g
• Potassium ferricyanide, 2.5g
• Distilled water, 200ml

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 is, however, an effective way of neutralizing this toxic compound.

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.

Description

A semi-permanent mounting media for immunofluorescence microscopy.

Method

1. Add the following reagents to a 250 ml flask or beaker:

• 24 g analytical grade glycerol (Sigma #G-6279)
• 9.6 g Mowiol 4-88 (Fluka, #81381, can be purchased through Sigma-Aldrich)
• 24 ml distilled water
• 48 ml 0.2M Tris buffer, pH 8.5

2. Stir with a clean stir bar on a hot plate on warm (not boiling)at least 4-5 hours until the majority of the Mowiol powder goes into solution.
3. Aliquot into 50 ml centrifuge tubes, weigh and balance
4. Centrifuge at 5000g for 15 minutes. Carefully remove the supernatant without disturbing the pellet at the bottom of the flask.
5. Aliquot into 15 ml conical tubes – add only 10 mls to each tube to allow for expansion with freezing.
6. Aliquots may be stored at -20C for 12 months. Store at room temperature no more than one month.
7. To use, warm solution to room temperature to eliminate bubble formation. use approximately 10 ul of mounting media for an 18 mm coverslip.
8. Allow slides to dry overnight at room temperature in a light-tight box.

Description

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)