Immunohistochemistry (using floating section)
Tae-Un Han, Ellen Sidransky
Published: 2023-08-01 DOI: 10.17504/protocols.io.6qpvr36yovmk/v1
Abstract
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This is an immunohistochemistry protocol used for evaluation of pathology in mouse brain.
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We successfully detected Gfap, Iba1, CD68, TH and alpha-Syn and phospho S129 alpha synuclein protein expression using this
protocol.
- In this protocol, floating sections are mounted on slides first before starting blocking step. In this way, large number of sections can
be stained more efficiently.
Steps
1.
Mount floating sections onto slides and dry for `0h 30m 0s` (<45 min), While section drying, PAP pen drawing line and dry `0h 5m 0s`.
2.
PBS rehydrate `0h 10m 0s`, Wash with PBS for `0h 5m 0s` three times.
3.
Blocking sol `500µL`, incubate `1h 0m 0s` at `Room temperature`.
4.
Replace buffer to primary antibody sol, incubate `0h 10m 0s` at `4°C`.
5.
Wash in PBST three times for `0h 5m 0s`, `0h 10m 0s` and `0h 5m 0s`.
6.
Secondary antibody sol, incubate for `1h 30m 0s` at `Room temperature`.
7.
Wash in PBS three times for `0h 5m 0s`, `0h 10m 0s` and `0h 5m 0s`.
8.
Mounting with Vectorshield mounting media
