Nov 16, 2023
Immuno Fluorescence staining of human FFPE (formalin fixed paraffin embedded) gut mucosal biopsy
- 1University of Chicago
Protocol Citation: Ran RZ Zhou 2023. Immuno Fluorescence staining of human FFPE (formalin fixed paraffin embedded) gut mucosal biopsy. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3jrnlk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
I use this protocol and it's working
Created: November 16, 2023
Last Modified: November 16, 2023
Protocol Integer ID: 91039
Keywords: gut, FFPE, IF, fluorescence, staining
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.
Guidelines
The human gut mucosal tissues was retrieved by colonoscopy. The size of tissues range from 1 x 1 x 1 mm, to 2 x 4 x 4 mm.
Smaller tissues were embedded in 1% agarose gel before FFPE.
Materials
1x Citrate buffer 200 ml
20 ml 10x Citrate buffer
180 ml distilled water
1x PBST 1000 ml
100 ml 10x PBS
0.5ml 10% Tween20
900ml distilled water
Permeabilization buffer 5 ml
125ul 10% TritonX-100
3.3 1x PBST
0.5ml 10% BSA
Blocking buffer 5 ml
2.5ml 10% BSA
2.5ml 1x PBST
Histoclear II (National diagnostics HS-202)
Histology grade ethanol (VWR 89370-084)
10xCitrate buffer (MillieporeSigma C9999)
10xPBS (FisherSci BP399500)
10%Tween20 (Teknova T0710)
10%TritonX (MillieporeSigma 93443)
10%BSA (Miltenyi Biotec 130-091-376)
Safety warnings
Boiling buffer, microwave and hotplate are used in the protocol. Please proceed with caution and use thermal gloves at these steps.
Ethics statement
The human intestinal tissue are obtained after patients' consents and approval from Institutional Review Board at the University of Chicago (IRB Number: 15573A). All the samples are processed for research use only.
Before start
Check the tissue block quality with H&E histology.
Deparaffinization
Deparaffinization
15m
Immerse tissue sections on slides in Histoclear II in a staining dish Room temperature 00:05:00 . 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.
15m
Rehydration
Rehydration
16m
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 00:02:00 . Repeat this step one more times with fresh ethanol.
4m
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 00:02:00 .Repeat this step one more times with fresh 70% ethanol.
4m
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 00:02:00 . Repeat this step one more times with fresh 50% ethanol.
4m
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 00:02:00 . Repeat this step one more times with fresh water.
4m
Target retrieval
Target retrieval
26m 30s
Place the slides to a slide basket. Immerse the tissue sections in the boiling water 100 °C .
30s
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.
15m
Move the slides from citrate buffer. Cool the tissues in distilled water dish Room temperature .
1m
Remove all the beakers from the hot plate.
Airdry the slides Room temperature .
5m
Draw a hydrophobic barrier around the tissue sections on the slides.
5m
Prepare the humidity chamber with the heated water from step 13.
Permeabilization
Permeabilization
22m
Place the slides in a humidity chamber. Add 1 x PBS to the tissues Room temperature .
2m
Tap off water. Add permeabilization buffer to the tissues Room temperature 00:10:00 . Repeat one more time with fresh permeabilization buffer.
20m
Blocking and primary antibody incubation
Blocking and primary antibody incubation
17h 30m
Tap off the buffer. Add blocking buffer to the tissues Room temperature .
1h
Prepare the primary antibody dilutes in 1 x PBST.
Tap off the blocking buffer. Add antibody dilutes to the tissues 4 °C Overnight .
16h
Tap off buffers. Immerse the slides in a staining dish with 1 x PBST Room temperature 00:10:00 on a horizontal shaker. Repeat the rinse for two more times.
30m
Prepare the secondary antibody dilutes in 1 x PBST.
Secondary antibody incubation and mounting
Secondary antibody incubation and mounting
1h 15m
Tap off PBST. Place the slides in the humidity chamber. Add antibody dilutes to the tissues Room temperature 01:00:00 .
1h
Tap off buffers. Immerse the slides in a staining dish with 1 x PBST Room temperature 00:05:00 on a horizontal shaker. Repeat the rinse for two more times.
15m
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.