Immuno Fluorescence staining of human FFPE (formalin fixed paraffin embedded) gut mucosal biopsy
Ran RZ Zhou
Disclaimer
This protocol is for research only.
Abstract
This protocol is to provide a detail instruction on the immuno fluorescence staining on human gut mucosal biopsy that has been preserved in FFPE and sectioned at 5 micron thickness. The fixation for the tissues was 24 hour at room temperature in 10% neutral buffered formalin. Target retrieval needs optimization is fixation, sectioning conditions change.
Before start
Check the tissue block quality with H&E histology.
Steps
Deparaffinization
Immerse tissue sections on slides in Histoclear II in a staining dish Room temperature 0h 5m 0s . Move the slide holder up and down x 5 times. Repeat this step two more times with fresh Histoclear II. There should be no visible paraffine remaining on the slide by the end of this step.
Rehydration
Move to another staining dish. Shake off Histoclear II from former step. Immerse tissue sections on slides in histology ethanol in a staining dish Room temperature 0h 2m 0s . Repeat this step one more times with fresh ethanol.
Move to another staining dish. Shake off ethanol from former step. Immerse tissue sections on slides in 70% histology ethanol in a staining dish Room temperature 0h 2m 0s .Repeat this step one more times with fresh 70% ethanol.
Prepare fresh 1 x Citra buffer 200 ml in a glass beaker, and place 200 ml distilled water in the second glass beaker.
Move to another staining dish. Shake off ethanol from former step. Immerse tissue sections on slides in 50% ethanol in a staining dish Room temperature``0h 2m 0s . Repeat this step one more times with fresh 50% ethanol.
Microwave the liquid in the beakers x 1 minute. Place the beakers with liquid on the hot plate and bring the liquid to the boiling point.
Move to another staining dish. Immerse tissue sections on slides in distilled water in a staining dishRoom temperature``0h 2m 0s . Repeat this step one more times with fresh water.
Target retrieval
Place the slides to a slide basket. Immerse the tissue sections in the boiling water 100°C .
Move the slides from boiling water. Immerse the tissue sections in boiling 1 x citrate buffer100°C . There should be no agarose gel remaining on the slides.
Move the slides from citrate buffer. Cool the tissues in distilled water dish Room temperature.
Remove all the beakers from the hot plate.
Airdry the slides Room temperature .
Draw a hydrophobic barrier around the tissue sections on the slides.
Prepare the humidity chamber with the heated water from step 13.
Permeabilization
Place the slides in a humidity chamber. Add 1 x PBS to the tissues Room temperature.
Tap off water. Add permeabilization buffer to the tissues Room temperature 0h 10m 0s . Repeat one more time with fresh permeabilization buffer.
Blocking and primary antibody incubation
Tap off the buffer. Add blocking buffer to the tissues Room temperature .
Prepare the primary antibody dilutes in 1 x PBST.
Tap off the blocking buffer. Add antibody dilutes to the tissues 4°C .
Tap off buffers. Immerse the slides in a staining dish with 1 x PBST Room temperature 0h 10m 0s on a horizontal shaker. Repeat the rinse for two more times.
Prepare the secondary antibody dilutes in 1 x PBST.
Secondary antibody incubation and mounting
Tap off PBST. Place the slides in the humidity chamber. Add antibody dilutes to the tissues 4Room temperature 1h 0m 0s .
Tap off buffers. Immerse the slides in a staining dish with 1 x PBST Room temperature 0h 5m 0s on a horizontal shaker. Repeat the rinse for two more times.
If nuclei staining is need, incubate tissues with DAPI for 2 minutes at room temperature between the second and third rinses.
Tap off buffer. Add prolong Gold to the tissues and cover with cover glass.
