Key Points:
BE SCIENTIFIC! It really is to your advantage in this
sort of stuff to be very careful to keep things constant, both
within and between experiments. So be quite stringent with temperatures,
timings and concentrations (especially of substrates and counterstains).
It could be particularly important in the microwave step where
many keep the buffer volume and slide number constant from experiment
to experiment (using blank slides and slide holders where necessary).
1. Dewax in Xylene. 2 X 10'
2. 100% Ethanol. 2'
3. 100% Ethanol. 2 X 1'
4. 95%, 80%, 70%, 50%, 30%. 1'@
5. PBS 5'
6. Block endogenous peroxidase activity using 3% H2O2 in methanol (or H2O)for 10'
followed by washing in H2O.
Endogenous alkaline phosphatase activity (but not phosphatase
activity)is completely blocked after 10' in the microwave I don't
know if this is true when one uses the wet autoclave treatment.(J.Histochem.Cytochem.
vol.43, no.1, pp. 97-102, 1995).
7. Rinse in distilled water 5'
8. PBS 5'
Brief wash in MW or AC buffer ?
9. Pretreat the sections with either wet autoclave or microwave.
Buffers: A variety of buffers have been used for this type of thing. (see Shi et al. J.Histochem.Cytochem. vol.43, no.2, pp. 193-201, 1995). pH seems to be important and the optimum varies from antibody to antibody. It seems that most react well to a slightly alkaline environment so this sort of buffer is a good one to start with.
Tris-HCL 0.1M (pH7.2-9.0 (use pH8))or Citrate buffers (Citric
acid, sodium citrate 0.1M (pH3.0-6.2))seem to be good.
Wet Autoclave pretreatment.
9a. Put the sections in heat resistant plastic Coplin jars (tubs or similar) filled with buffer and put the lid on (not air tight). Autoclave (in the small AC) for 10 minutes at120oC.
When the cycle is finished remove the slides at about 80 oC and the let them cool to room temperature.
The treatment seems to vary for different antibodies and so
5' at 120oC may be better. A similar situation occurs with the
microwave pretreatment and so it must be worked out empirically
what the best conditions are.
Microwave pretreatment.
9b. Put the sections in heat resistant plastic Coplin jars (tubs or similar) filled with buffer (at least 300ml) and put the lid on (not air tight). Make sure that you have removed any metal bits! Heat twice on maximum power (600-800W) for 5 minutes each with 1 minute cooling in between (also top up the buffer, if need be, to prevent sections drying out).
When the heating is finished remove the slides and the let them cool to room temperature.
Again the exact timing for any particular antibody should be
worked out empirically but a ten minute dip in the MW is a pretty
good starting point for many antibodies. Some antibodies still
perform well after as much as 25-30 minutes total microwave treatment
so there is many possibilities as far as multiple staining goes
(J.Histochem.Cytochem. vol.43, no.1, pp. 97-102, 1995).
10. ddH2O 2'
11. PBS 5'
12. Incubate with serum (diff. if m/c or p/c antibody) 20' at 37oC
13. Tap off serum and wipe away excess.
14. Incubate with 1o antibody, diluted in PBS. o/n at 4 oC then 20' at RT
then 20' at 37oC
15. Tap off 1o antibody and wash in PBS. 5' x 2
16. Incubate with biotinylated 2 o ab. 30' at 37oC
17. Tap off 2o antibody and wash in PBS. 5' x 2
18. Incubate with ABC complex/HRP. 30' at 37oC
19. Tap off ABC complex/HRP and wash in PBS. 5' x 2
20. Immerse in TBS. 5'
(TBS = ?)
21. Incubate with peroxidase substrate. 3-10' (use 5' first) RT
(substrate = DAB 0.05%, H2O2 0.03% in TBS).
22. Counterstain and mount with coverslip.