Immunofluorescence, Sirius red staining, and intensity quantitations

Immunofluorescence 

1. Flash freeze dissected wound tissue in O.C.T. in a cold 100% ethanol bath over dry ice then store at -80^oC. 

2. Cryosection tissue into 12-14μm thick sections and store at -80^oC.

3. Thaw sections, taking care that tissue never dries. Fix with cold 4% PFA/PBS for 5 minutes.

4. Wash slides 3x in PBS and pre-block 10 minutes (see block solutions below)*.

5. Incubate tissue with antibody in appropriate block solution  at 4^oC for recommended time (see Table I). Fluorescein- or biotin-conjugated antibodies should be used whenever possible to avoid non-specific staining by secondary reagents.

6. Wash slides 3x and incubate in secondary reagent if required.

7. Fix tissue with cold 4% paraformaldehyde for 5-10 minutes.

8. Mount in Vectashield DAPI (Vector H-1200) and examine within 48 hours (slides may be stored at 4^oC).

* To identify nuclear localization of b-Catenin, permeabilize tissue with 0.3% Triton X/PBS for 2 minutes, then wash 3x before pre-block.

Blocking and antibody dilution buffers:

1. For examination of ECM and cell surface proteins: PBS containing 1% BSA, 1:10 anti-mouse CD16/CD32 Fc block (eBioscience). 

2. For examination of internal cellular proteins: PBS containing 1.25% cold water fish gelatin, 0.05% saponin.

Table I. Antibodies.

* overnight

Sirius Red staining

1. Thaw 20μm thick frozen tissue sections without allowing tissue to dry. 

2. Fix tissue for 1 hour in 4% PFA/PBS. 

3. Wash twice briefly in water.

4. Apply picro-sirius red solution evenly over each slide. Leave for exactly 1 hour at room temperature. (It is important that all slides are treated for the same length of time as staining intensity will continue to increase.) 

5. Wash twice with acidified water.

6. Dry slide until tissue is almost dry. Do not over-dry.

7. Mount rapidly in solvent-based (resinous) mounting medium. 

Solutions:

1. Picro-sirius red: 0.5 g Direct-Red 80 (Sirius red F3BA, Sigma-Aldrich cat. #36-554-8) in 500ml saturated aqueous picric acid solution. Store at RT.

2. Acidified water: 5 ml glacial acetic acid in 1 liter water. 

For additional information, see http://www.ihcworld.com/_protocols/special_stains/sirius_red.htm 

Fluorescence or colorimetric intensity quantitations using Imagej. 

High resolution images of fluorescein- or dye-stained samples, acquired under identical conditions, can be compared for relative fluorescence or colorimetric (Sirius red) intensity using Imagej.

1. In Imagej, set scale of image based on known parameters (eg scale bar) (Analyze>Set Scale)

2. Change image to “grayscale”.

3. Adjust image threshold (Image>Adjust>Threshold).

4. Set measurements to be analyzed (Analyze>Set Measurements: check "Area", "Area Fraction", "Limit to Threshold" and "Display Label". 

5. Select background and test regions within image using rectangle tool. 

6. Press "m" (Analyse>Measure). The area and percent area will be displayed in the "Results" window.

7. CTCF values can be calculated as follows:

CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings)

For additional information, see 

https://imagej.nih.gov/ij/docs/examples/stained-sections/index.html, https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html

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