Appendix A- Autofluorescence

Problem tissues:

• epithelia (stratum corneum & glandular)

• leukocytes (neutrophil and eosinophil)

• Myocardial fibers

• Nervous tissues

Causes:

• endogenous enzyme

• double bonding

• aldehyde fixation

• age

Fixatives:

1. methanol with 1% acidic acid & 1% Na nitroferricyanide

2. 0.05% phenylhydrazine

3. H2O2

4. 0.074% HCl in absolute ETOH (0.2 ml/100ml).

Quenching procedures for autofluorescence

1. Formaldehyde vapor (60oC - 80oC)

2. Eriochrome black

3. Sodium nitrite

4. 1:5 or 1:20 IgG anti-sera

5. Pronase

6. Trypsin

7. Pepsin (4mg/ml in 0.01 N HCl @ 37oC for 20-30 minutes

8. Mayer’s hematoxylin counterstain

9. Pontamine Sky Blue

Appendix B- Dot-Blot Test

1. Nitrocellulose paper is cut into narrow strips. Use forceps to handle paper.
2. Wash the paper 5 minutes by gentle agitation in distilled water.
3. Let dry at room temperature.
4. A diluted solution or suspension of antigen in saline is dotted onto the filter. Use ~20ul.

• Complex antigens can be used at 0.1-1.0mg/ml.
• Less complex antigens can be diluted accordingly.
• Nucleic acid binding requires baking 60 minutes at 80oC.

5. Dry thoroughly. Can be stored several weeks at this stage.
6. Blocking is done with 3% BSA in 1% normal serum in TBS with gentle agitation for 15 minutes.
7. Incubate with the test antibody for 2-4 hours at room temperature, or overnight.
8. Wash in TBS 30 minutes (several changes).
9. Repeat the blocking step.
10. Incubate the filter for 2 hours with a horseradish peroxidase-conjugated secondary anti body targeted against the test antibody. Concentration may vary, but 1/1000 in blocking solution is about right.
11. Wash in TBS 30 minutes (several changes).
12. Develop with 4-chloro-1-naphthol and hydrogen peroxide.
13. A positive reaction results in a blue spot on a white background in 2-15 minutes.

This test can be used to determine optimum concentrations and fixatives before actual immunostaining of experimental samples.

Development solution:

3mg/ml chloronaphthol in methanol. Stock solution can be kept in the dark, in the cold, for 1-2 weeks. Discard when signs of yellowing appear. Just before use dilute with 5 volumes TBS and 0.01% hydrogen peroxide.

Appendix C- Sample Protocol for Immunofluorescent Staining of Frozen Sections

1. Cut 8-10mm sections and place on a Super Frost Plus charged slide. Allow slide to come to room temperature and dry. *
2. Block for non-specific proteins by placing slides in a solution of PBS (pH 7.4) with 10% normal serum (of the animal the secondary antibody was made in). Block for 30 minutes at room temperature.
3. Place in primary antibody diluted with PBS (pH 7.4) with 1% fetal bovine serum.** This step may be done for 1 hour at room temperature or overnight at 4oC.
4. Wash well in 3 changes of PBS @ 5 minutes each (with agitation if possible).
5. Apply secondary antibody (against animal of primary antibody and conjugated with a fluorescent probe) diluted with PBS (pH 7.4) with 1% fetal bovine serum. Leave in this solution for 30 minutes.
6. Wash well in 3 changes of PBS @ 5 minutes each (with agitation if possible).
7. Mount with Vecta Shield mounting media. Seal the edges with nail polish. If slides will not be viewed immediately, store in a humid, dark, and cool environment for up to a week.

* This is the only time the samples should be allowed to dry out. After step two they must remain moist, or false positives may result.
** Both primary and secondary antibody dilutions must be determined by running a series of dilution factors for each. Do this even if the antibody has been tested in another laboratory. The following chart may be helpful.

Slide # Primary dilution Secondary dilution Amount of specific staining(++,+,-)

Appendix D- Standard Incubation Schedule for EM-Immunogold Post Embedment Staining

Prepare Incubation Buffer:

PBS, (10mM Phosphate buffer, 150 mM NaCl), pH 7.4

0.5% Bovine Serum Albumin, 0.1% gelatin, 20mM NaN3. Check the pH and adjust to 7.4 if necessary.

1. Incubate with 0.05 M glycine or lysine in PBS:15 minutes


2. Block in 5% normal serum (same species as secondary): 30 minutes


3. Incubate with a dilution of the primary antibody (1-5ug/ml) in incubation buffer or overnight at 4oC: 30 minutes-1 hour


4. Incubation buffer: 6 x 5 minutes


5. Dilution of Immunogold conjugate (1/75-1/150): 2 hours


6. Incubation buffer: 6 x 5 minutes


7. PBS: 3 x 5 minutes


8. Post-fix in 2.5% glutaraldehyde: 5 minutes


9. PBS: 5 minutes


10. ddH2O
: 5 x 2 minutes


11. Contrast stain as usual

All solutions are placed as droplets on clean parafilm surface. Grids are placed, section down on top of droplet for incubation times.

Do NOT use copper grids. Nickel grids are preferred as they will not react with gold colloid.

Appendix E- Epitope Retrieval

A. Heat Induced epitope retrieval (HIER)

Buffers

• 1mM EDTA pH 8.0

• 0.01M Citrate buffer pH 6.0

• Tris-HC

• Urea

• Heavy metal solutions (zinc, lead)

• Other company-made products

Heat solution to a boil using pressure cooker, rice steamer, hot plate, hydrated autoclave or microwave. Place sections in solution and continue heating 10-30 minutes.

B. Enzyme epitope retrieval

Common enzymes used for this purpose

• Trypsin

• Pepsin

• Pronase

• Protein Kinase*

• Ficin

• Dnase

C. Ultrasonic

D. Formic acid