VECTOR Laboratories Protocol
Note: For JCB paper, Nasrin used 20 uM embryo sections and 3 or 4 sections per
poly-L-lysine coated slide.
SUPPLIES:
Vectastain Elite ABC Kit: Universal is good kit if 10 antibody is rabbit. (Vector Cat. #
PK-6200)
DAB perioxidase substrate kit (Vector Cat. # SK-4100)
Hematoxlin QS (Vector Cat. # H-3404)
VECTASHIELD mounting medium for fluorescence (Vector Cat. # H-1000)
- a Vectashield with DAPI is also available.
Prepare VECTASTAIN working solutions:
1. 10mM sodium phosphate, pH 7.5, 0.9% saline (PBS)
2. Blocking serum*:
- 5 mL of buffer in mixing bottle (yellow label)
- add 1 drop (50uL) stock (yellow label)
* Preferred serum for blocking is from the same species in which the biotinylated 20
antibody is made.
0
3. Biotinylated Universal Antibody (2 Antibody):
- 5 mL buffer in mixing bottle (blue label)
- add 2 drops (100 uL) of normal blocking serum stock (yellow label)
- add 2 drops (100 uL) of biotinylated antibody stock (blue label)
4. Vectastain Elite ABC Reagent (ABC Complex
- 5 mL buffer in ABC Reagent large mixing bottle
- 2 drops of REAGENT A (gray label)
- MIX IMMEDIATELY
- LET STAND 30 MINUTES BEFORE USE
Reagent):
5. DAB:
- To 5 mL water add 2 drops buffer stock solution.
- Add 4 drops DAB stock solution; MIX WELL.
- Add 2 drops H2O2 solution; MIX WELL.
Optional: For a black/gray stain add to the DAB solution 2 drops Nickel solution and
mix well.
1. Sections are air dried.
2. Fix sections with acetone or appropiate fixative for the antigen under study.
3. Transfer slides into buffer for 5 minutes.
4. Incubate sections for 20 minutes with normal blocking serum.
5. Blot excess serum from sections.
6. Incubate sections for 30 minutes with diluted biotinylated 10
diluted in buffer.
antibody
Note that 10 antibodies are 20 to 100 X more conc. than for a western blot.
7. Wash slides for 5 minutes in buffer.
8. Incubate sections for 30 minutes with diluted biotinylated 20
solution.
antibody
9. Wash slides for 5 minutes in buffer.
10. Incubate for 30 minutes with VECTASTAIN Elite ABC Reagent
(ABC
Complex Reagent).
11. Wash slides for 5 minutes in buffer.
12. Incubate sections @ RT in peroxidase substrate solution (DAB) until
desired intensity develops (2 to 10 minutes).
13. Wash slides for 5 minutes in water.
14. Counterstain.
Counterstain Method 1:
1. Immerse slides in Vector Hematoxylin QS (blue-violet nuclear staining)
counterstain or apply counterstain directly to slide, completely covering the
section.
2. Incubate sections with Vector Hematoxylin QS for 5 to 45 seconds.
3. Rinse sections with running tap water until rinse water is colourless or dip for
approximately 10 seconds in water.
4. Add mounting media and coverslip.
ADD COVERSLIP:
5. Special pipet dispenses 1 drop (25 uL) onto the section which is suitable for a
22x22 mm coverslip.
6. Let slides dry for at least 24 hours.
Slides are good for a few weeks. For long-term, permanately seal the perimeter with nail
polish or plastic sealant. Store mounted slides @ 4 0C.
Counterstain Method 2:
Make Hematoxylin:
Hematoxylin
Ammunoium alum [(NH4)2SO4 Al2(SO4)3-24H2O]
Ethanol
Glycerol
H2O
6.4g
60g
200 mL
160 mL
640 mL
Mix for 2 to 4 hours. Allow stain to ripen in dark for 6 to 8 weeks before using. it will
work immediately after mixing, but resulting stain will be dull.
7. Apply Hematoxylin directly to section for 2 minutes. (NUCLEAR STAIN)
8. Quickly move to tap water to wash.
9. Distilled water for 5 to 10 dunks.
10. 1% acid alcohol (70mL 70% EtOH + 0.7 mL conc. HCl) for 10 dunks.
11. Tap water until sections are blue (10 to 15 minutes).
12. Distilled water for 5 to 10 dunks.
13. Eosin for 7 dunks. (CYTOPLASMIC STAIN)
14. Move to tap water to wash.
15. Distilled water for 5 to 10 dunks.
16. 70% EtOH for 60 seconds.
17. 95% EtOH for 60 seconds.
18. 100% EtOH for 2 minutes.
19. 100% EtOH for 2 minutes.
20. Xylene for 5 minutes.
21. Xylene for 5 minutes.
- Remove slide only when ready to quickly cover with coverslip.
- Save xylene in fumehood for re-use.
ADD COVERSLIP:
22. Special pipet dispenses 1 drop (25 uL) onto the section which is suitable for a
22x22 mm coverslip.
23. Let slides dry for at least 24 hours.
Slides are good for a few weeks. For long-term, permanately seal the perimeter with nail
polish or plastic sealant. Store mounted slides @ 4 0C.
EXTRA INFO. …
DAB
- diamino-benzidine tetrahydrochloride
- localizes perioxidase in tissue sections
- insoluable in alcohol and clearing agents allowing sections to be
perminately mounted
- intense purple precipitation; perhaps reddish-brown in sections.
Hematoxylin QS
- blue-violet nuclear staining
VECTASHIELD mounting medium
- aqueous.