You wrote :
MK>I plan to perform immunocytochemistry on fresh brain biopsy material.
MK>Stromal mucopolysaccharides may interfere. Any suggestions, i.e,
MK>cell pre-treatments, staining protocols, etc., to deal with this?
We are doing immunohistochemistry in the enteric nervous system
(submucosal plexus,myenteric plexus) maybe you can use our protocol it
is very easy to do :
1) You need tissue in slices thin enough to visualize under
microscope.
2) Fix the tissue in "Zamboni Fixative pH 7.3 1000 mL"
How to make Zamboni :
a) 20 g paraformaldehyd
b) 150 ml dobblefiltered picric-acid
c) Heat to 60 C, and adjust pH with NaOH to 7.3
d) Filter and add PhosphateBuffer (PB)
How to make PB (1000 mL)
a) 22.45 g Na2HPO4,2H20
b) 3.3 g NaH2PO4,H2O
c) destill water - fill up to 1000 mL
3) The tissue stays in Zamboni overnight at 4 Celcius.
4) Wash tissue in Phosphate Buffered Saline PBS
Wash (e.g. on a rotating table)
10 min in each of in following solution
50 % alcohol
80 % alcohol
95 % alcohol
100 % alcohol
Xylene
100 % alcohol
95 % alcohol
80 % alcohol
50 % alcohol
How to make PBS
a) 8.5 g NaCl
b) 1.070 g Na2HPO4
c) 0.345 g NaH2PO4,H20
d) fill up with destill water to 1000 ml
e) solution adjust to pH 7.1 with 0.2 M NaH2PO4
How to make 0.2 M NaH2PO4
6.899 g NaH2PO4,H2O to 250 mL destil H20
f) some people add 0.1 % Triton X-100
5) After wash in alcohol and Xylene wash 3x10 min in PBS
6) Store tissue at 4 Celcius in PBS
7) Immunohistochemistry against VIP (example from my lab)
a) A slice of tissue (1 cm²) is spread on micros.slide
b) Add 50 µL primary antibody (1:25)
How to make primary antibody
a) Buy a kit at e.g. Peninsula
b) dissolve in destil water
instructions in the manual
from the kit
c) Make prober dilutions
Try with different levels
e.g. 1:25 and 1:50 and 1:100
No staining means to diluted
c) Tissue stay overnight in primary antibody
d) Add secondary antibody (e.g. Texas Red)
How to make secondary antibody
a) dissolve in destil water
c) Make dilutions
Try 1:10, 1:25
If there is color all over the
tissue the secondary was
probably to strong.
e) Tissue stay 2 hours in secondary antibody
8) Get a microscope with the right filter and look at it
fast, it will probably fade fast. Some tricks exist to
keep it from fading. Sometimes the color is gone after
1-2 days
9) Best to do all your antibody procedures in complete
dark.
Best Regards
MSc Lars Thomsen 02-08-95 03.35
Royal Veterinary & Agricultural University
Copenhagen, Denmark
EMAIL : lars.thomsen at dkb.dk
FAX : +45 35 38 25 25
TLF : +45 35 28 25 13
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