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Protocols
cryosectioning,
avidin-biotin, sample fixation |
Buffers
tissue fixation, permeabilization,
PBS ... |
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Links
to
microscopy and histochemistry resources
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Immunohistochemistry reagents
and important buffers are listed here. There are also commercially
available immunohistochemistry kits and protease inhibitor cocktails.
It is important to protect
antibodies from freeze-thaw effects by proper aliquoting.
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Phosphate
Buffered Saline - pbs |
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8
g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4
800 ml dH2O
pH - 7.4
dH2O up to I liter
autoclave
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| Citrate
Buffer |
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2.1
citrate monohydrate
1000ml distilled H2O
adjust pH to 6.0
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| DAB |
5
ml dH2O
2 drops stock buffer
4 drops stock DAB
2 drops H2O2 (hydrogen peroxide)
filter using syringe-based system
add DAB to 200ml PBS
note:
- prepare DAB in darkened room
- DAB is believed to be carcinogenic
- after DAB is used, add bleach to solution prior
to discarding
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| Endogenous
peroxidase quenching buffer |
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200ml
PBS
50 ml 30% H2O2
mix and use immediately
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| Mayer's
Hematoxylin |
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1000ml
dH2O
aluminum potassium alum 50g
5 g hematoxylin powder
0.4g sodium iodate
20ml glacial acetic acid
dissolve
alum in dH2O
dissolve hematoxylin into solution
add sodium iodate
add glacial acetic acid
bring solution to a boil, remove from heat and
allow to cool
filter using gravity filtration
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| Paraformaldehyde
(PFA) |
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dissolve
4 g PFA in 50ml dH2O
add 2 drops 10N NaOH
add a stir magnet; stir and heat to 60 C - until
solution is clear
add 50ml 2x PBS
adjust pH to 7.3
PFA
should be made fresh as needed |
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