Staining Protocols for Lymphoid Tissue: Technovit Glycol Methacrylate: Technical Information and Help
From Heraeus Kulzer.
Normal immunostaining procedures for routine markers on
sections obtained from lymphoid tissue. Procedure:
- Dry the sections for 2 hours
at 37�C on a slide warmer. This
must be done whether the slides have been stored at 4�C
for some time or just collected.
- Pretreat the sections with trypsin
at 37�C. Trypsin concentrations
must be determined separately for each antibody. Use trypsin
solution that has been preheated at 37�C for 30
minutes. Cover section with at least 100 microliters
of solution.
- Wash in PBS for 10 minutes at room temperature. Refresh the
buffer 4 to 5 times.
- Preincubate in normal serum
from the animal species in which the second antibody
is raised for 30 minutes at 37�C. Only
perform this step if aspecific background
is present using a particular antibody.
- Drip off excess serum and apply
the first antibody in an appropriate concentration
and incubate for 2 hours at 37�C.
- Wash in PBS for 10 minutes at room temperature. Refresh the
buffer 4 to 5 times.
- Block endogenous peroxidase in a solution of 0.06% hydrogen
peroxide in phosphate buffered saline, pH 7.4,
for 30 minutes at room temperature.
- Wash in PBS for 10 minutes at room temperature. Refresh the
buffer 4 to 5 times.
- Incubate in appropriate dilutions of the secondary antibody,
containing 5% normal serum for 60 minutes at
room temperature.
- Wash in PBS for 10 minutes at room temperature. Refresh the
buffer 4 to 5 times.
- Develop the peroxidase activity in daminobenzidine (DAB).
- Counterstain the sections in hematoxylin or,
if a more advanced morphological detail is
necessary, in periodic-acid-Schiff reagent.
- Cover with glycerin-gelatin and cover glass
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