Immunostaining on Paraffin Sections of Fly Heads

Fix fly heads overnight in 4% formalin.

Embed in paraffin.

Cut 4 micron sections.  Dry at 42°C .

Deparaffinize and bring through ethanols to water (xylene x 2, 100% ethanol x 4, tap H₂O x 2).

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.