Immunostaining of H&E Stained Paraffin Sections of Fly Heads
Mel Feany
Abstract
This protocol describes how to perform immunostaining on H&E stained paraffin sections of fly heads.
Steps
Embed the fly heads in paraffin and stain with H&E for neuronal counting, per the following protocol:
dx.doi.org/10.17504/protocols.io.4r3l24on4g1y/v1
Microwave slides in 10millimolar (mM) sodium citrate for 0h 15m 0s.
Cool 0h 20m 0s .
Stock: 100millimolar (mM) sodium citrate, pH 6.0
Use at least 1L of citrate solution in large glass box to avoid drying.
Block in PBST (PBS with 0.3% Triton) with 2% dry milk for 0h 30m 0s
Stock: 10X PBS
Incubate with primary antibody at Room temperature .
Wash 3 x in PBST.
Incubate with appropriate biotinylated secondary (for immunohistochemistry) or fluorescent secondary (for immunofluorescence) antibody at 1:200 in PBST + milk for 1h 0m 0s at Room temperature .
For immunohistochemistry incubate in ABC reagent (Vector) for 1h 0m 0s at Room temperature .
Rinse 3 x PBST.
Mount slides with antifading medium for immunofluorescence or dehydrate through ethanol series and xylenes and mount in Permount for immunohistochemistry.
