This is adapted from sodium bromide preparations of human scalp specimens which yields individual hairs (J Invest Dermatol 1996 Jan;106(1):28-35). I used it to examine epidermal melanocytes but the ENTIRE hair shaft and its appendage can be viewed in its entirety (Very cool!). About 2/3 of the anagen phase hairs remain in the dermis side and are more difficult to visualize but I am trying to clear the tissue to see if this will help. The DOPA rxn has to be done first; something in the beta-gal staining buffer inactivates the DOPA -- maybe the cyanates. The same salt-split technique can be used in whole-mount immunohistochemistry which I have done with the PEP8 (TRP2) antibody from V.Hearing. I can send you this protocol as well. The key is that instead of using DAB or chloronapthol I use AEC as my chromogen. The reason is that you can bleach out pigment (hairs etc.) in 10% H2O2 in PBS or water O/N and it wouldnt effect the red signal of the AEC.
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1. Isolate tail or footpad skin (Shave or snip excise)
2. Place in 2M (~20%w/v) Sodium Bromide 1ml for single specimens (24-well) or batch prep in 5-10ml
3. Incubate at room temperature for 3hr to O/N (for DOPA, X-gal stains, I usually only do 3-4hrs; O/N and 4C is okay for immunohistochemistry).
4. Separate epidermis from dermis with forceps (should separate easily otherwise not ready).
5. Wash both epidermis and dermis halves in 0.1 M phosphate buffer pH 6.8; repeat
6. Incubate in 50mM DOPA at 37C x 3hrs, protect from light. (Again, if multiple samples, this can be done with 1ml in a 24-well)
7. Check staining by placing tissue on glass slide, uncovered under microscope. Overstaining leads to increased particulate deposition which is difficult to clean off.
8. Wash tissue in PBS x 2, briefly fix in 1% or 4% PFA x 15min. Wash in PBS.
9. Incubate O/N in Beta-galactosidase staining buffer (1 mg/ml X-gal, 5 mM potassium ferricyanate, 5 mM potassium ferrocyanate, 2 mM MgCl2, 0.2% Non-idet-P40, and 0.1% sodium deoxycholate in PBS).
10. Re-fix tissue; can photograph whole mounts or clear in glycerol gradients.
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ORDERING INFORMATION:
1. Sodium Bromide Sigma S9890; 20.6g/100ml in H2O = 2M
2. L-DOPA Sigma D9628; 50mg/100ml in 0.1 M phosphate buffer pH 6.8 = 50mM
3. Beta-galactosidase staining buffer (1 mg/ml X-gal, 5 mM potassium ferricyanate, 5 mM potassium ferrocyanate, 2 mM MgCl2, 0.2% Non-idet-P40, and 0.1% sodium deoxycholate in PBS
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1. Isolate tail or footpad skin (Shave or snip excise)
2. Place in 2M (~20%w/v) Sodium Bromide 1ml for single specimens (24-well) or batch prep in 5-10ml
3. Incubate at room temperature for 3hr to O/N (for DOPA, X-gal stains, I usually only do 3-4hrs; O/N and 4C is okay for immunohistochemistry).
4. Separate epidermis from dermis with forceps (should separate easily otherwise not ready).
5. Wash both epidermis and dermis halves in 0.1 M phosphate buffer pH 6.8; repeat
6. Incubate in 50mM DOPA at 37C x 3hrs, protect from light. (Again, if multiple samples, this can be done with 1ml in a 24-well)
7. Check staining by placing tissue on glass slide, uncovered under microscope. Overstaining leads to increased particulate deposition which is difficult to clean off.
8. Wash tissue in PBS x 2, briefly fix in 1% or 4% PFA x 15min. Wash in PBS.
9. Incubate O/N in Beta-galactosidase staining buffer (1 mg/ml X-gal, 5 mM potassium ferricyanate, 5 mM potassium ferrocyanate, 2 mM MgCl2, 0.2% Non-idet-P40, and 0.1% sodium deoxycholate in PBS).
10. Re-fix tissue; can photograph whole mounts or clear in glycerol gradients.
__________________________________________________________________
ORDERING INFORMATION:
1. Sodium Bromide Sigma S9890; 20.6g/100ml in H2O = 2M
2. L-DOPA Sigma D9628; 50mg/100ml in 0.1 M phosphate buffer pH 6.8 = 50mM
3. Beta-galactosidase staining buffer (1 mg/ml X-gal, 5 mM potassium ferricyanate, 5 mM potassium ferrocyanate, 2 mM MgCl2, 0.2% Non-idet-P40, and 0.1% sodium deoxycholate in PBS