TITLE: Immuno Fluorescence staining of human FFPE (formalin fixed paraffin embedded) gut mucosal biopsy
AUTHORS: Ran RZ Zhou

[DESCRIPTION]
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.

[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.

[STEPS]
SECTION: Deparaffinization
1. Immerse tissue sections on slides in Histoclear II in a staining dish Room temperature 5 min . 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.

SECTION: Rehydration
2. 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 2 min  . Repeat this step one more times with fresh ethanol.

SECTION: Rehydration
3. 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 2 min .Repeat this step one more times with fresh 70% ethanol.


SECTION: Rehydration
None. Move to another staining dish. Shake off ethanol from former step. Immerse tissue sections on slides in 50% histology ethanol in a staining dishRoom temperature. 
SECTION: Rehydration
None. Move to another staining dish. Shake off ethanol from former step. Immerse tissue sections on slides in 50% histology ethanol in a staining dish Room temperature. 
SECTION: Rehydration
4. Move to another staining dish. Shake off ethanol from former step. Immerse tissue sections on slides in 50% ethanol in a staining dish Room temperature2 min . Repeat this step one more times with fresh 50% ethanol.

SECTION: Rehydration
5.  Move to another staining dish. Immerse tissue sections on slides in distilled water in a staining dishRoom temperature2 min . Repeat this step one more times with fresh water.

SECTION: Target retrieval
6. Place the slides to a slide basket. Immerse the tissue sections in the boiling water 100 °C .

SECTION: Target retrieval
7. 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. 

SECTION: Target retrieval
8. Move the slides from citrate buffer. Cool the tissues in distilled water dish Room temperature. 
SECTION: Target retrieval
9. Airdry the slides Room temperature . 

SECTION: Target retrieval
10. Draw a hydrophobic barrier around the tissue sections on the slides.
SECTION: Permeabilization
11. Place the slides in a humidity chamber. Add 1 x PBS to the tissues Room temperature.

SECTION: Permeabilization
12. Tap off water. Add permeabilization buffer to the tissues Room temperature 10 min . Repeat one more time with fresh permeabilization buffer.

SECTION: Blocking and primary antibody incubation
13. Tap off the buffer. Add blocking buffer to the tissues Room temperature .

SECTION: Blocking and primary antibody incubation
14. Tap off the blocking buffer. Add antibody dilutes to the tissues 4 °C   .

SECTION: Target retrieval
8.1. Remove all the beakers from the hot plate.
SECTION: Blocking and primary antibody incubation
13.1. Prepare the primary antibody dilutes in 1 x PBST.
SECTION: Target retrieval
10.1. Prepare the humidity chamber with the heated water from step 13.
SECTION: Blocking and primary antibody incubation
15. Tap off buffers. Immerse the slides in a staining dish with 1 x PBST Room temperature 10 min  on a horizontal shaker. Repeat the rinse for two more times.

SECTION: Rehydration
3.1. Prepare fresh 1 x Citra buffer 200 ml in a glass beaker, and place 200 ml distilled water in the second glass beaker.
SECTION: Rehydration
4.1. 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. 
SECTION: Secondary antibody incubation and mounting
16. Tap off PBST. Place the slides in the humidity chamber. Add antibody dilutes to the tissues 4 Room temperature 60 min .
SECTION: Blocking and primary antibody incubation
15.1. Prepare the secondary antibody dilutes in 1 x PBST.
SECTION: Secondary antibody incubation and mounting
17. Tap off buffers. Immerse the slides in a staining dish with 1 x PBST Room temperature 5 min  on a horizontal shaker. Repeat the rinse for two more times.
SECTION: Secondary antibody incubation and mounting
17.1. If nuclei staining is need, incubate tissues with DAPI for 2 minutes at room temperature between the second and third rinses.
SECTION: Secondary antibody incubation and mounting
18. Tap off buffer. Add prolong Gold to the tissues and cover with cover glass.