Caution: Formaldehyde is toxic and should be handled with caution under a chemical fume hood. Consult Material Safety Data Sheets for proper handling of all laboratory chemicals.

4% Paraformaldehyde (PFA)

1. Heat 250 mL of double strength phosphate buffer stock solution (see step 4) to 140癋 (60癈) in a beaker with a disposable stir bar in a hood.
2. Add 20 g granular paraformaldehyde and stir until it is dissolved.
3. Add 250 mL deionized water and filter the solution into a container placed on ice. The solution is ready when cold. Adjust pH to 7.0?.4.
4. Double Strength Phosphate Buffer Stock solution is prepared by dissolving 7.7 g NaOH and
   33.6 g NaH₂PO₄ in 1 liter deionized water.

4% Paraformaldehyde with 2% Gluteraldehyde.

1. Heat 250 mL double strength phosphate buffer stock solution (see above) to 140癋 (60癈) in a beaker with a disposable stir bar.
2. Add 20 g granular paraformaldehyde and 10 g gluteraldehyde and stir until it is dissolved.
3. Add 250 mL deionized water and filter the solution into a container placed on ice. The solution is ready when cold. Adjust pH to 7.0?.4.

Buffered Formaldehyde (Formalin)

1. Dissolve 32.5 g Na₂HPO₄ and 20 g NaH₂PO₄ in 4.5 L deionized water.
2. Add 500 mL 40% Formaldehyde.
3. Mix; Adjust pH to 7.0?.4.

Bouin抯 Fluid

1. Picric Acid (standard aqueous solution) 75 mL.
2. Formalin (40% aqueous Formaldehyde) 20 mL
3. Glacial Acetic Acid 5 mL.
4. Mix

Carnoy抯 Fixative

1. 10 mL of glacial acetic acid
2. 30 mL of chloroform
3. 60 mL of absolute alcohol (100% Ethanol)
4. Mix

PLP (Periodate-lysine-paraformaldehyde) Fixative

1. Dissolve 7.3 g of lysine monohydrochloride in 200 mL of ddH₂O.
2. Adjust pH to 7.4 with 0.1 M Na₂HPO₄ (Na₂HPO₄? H₂O 17.8 g/L) NOT phosphate buffer!
3. Complete volume to 400 mL with 0.1 M phosphate BUFFER (!!) pH 7.4. This lysine-phosphate buffer keeps for 2? days in the refrigerator, but can be frozen in aliquots for longer storage.
4. Just before use, mix 375 mL lysine-phosphate buffer with 100 mL 20% Formaldehyde and top to 500 mL with ddH₂O. Add 1.06 g Sodium periodate (NaIO₄ ) and mix well. The PLP fixative must be used within a maximum of 2 hrs.

Final concentrations: Lysine 75 mM, Formaldehyde 4%, Sodium periodate 10 mM. (Note: Some PLP formulations in literature also use 2% paraformaldehyde)

Acetone/Methanol Fixative

1. 100 mL acetone
2. Add 100 mL methanol
3. Mix well. Use fresh. 50?0 solution is used at room temperature or -20癈

APPENDIX B
Making Serial Antibody Dilutions

Reagents/Equipment:

• PBS or other appropriate buffer.
• Small capped tubes
• Pipets capable of accurate delivery of 200 mL and 1000 mL volumes

Keep buffer and tubes in ice

1. Pipet 450 礚 buffer into a tube.
2. Add 50 礚 antibody solution, and mix. This gives a 1:10 dilution of the antibody.
3. Label tubes A through M for 1:50, 1:100, 1:200, 1:400, etc. to 1:51,200 dilutions.
4. Pipet 1600 礚 of dilution buffer into tube A (to become a 1:50 dilution). Pipette 1000 礚
   (1.0 mL) of dilution buffer into tubes B through M (to become 1:100 - 1:102,400 dilutions).
5. Pipette 400 礚 of 1:10 antibody dilution into tube A (which contains 1600 礚 buffer). Mix well. This results in a 1:50 antibody dilution.
6. Take 1000 礚 of antibody sample from Tube A and add to Tube B (which contains 1000 礚 buffer). Mix well.
7. Take 1000 礚 of antibody sample from Tube B and add to Tube C (which contains 1000 礚 buffer), etc. Mix well.

APPENDIX C Protein A/G Binding Affinities

APPENDIX D
Enzyme Substrates for ELISA Testing (soluble substrates) and Blotting (insoluble substrates)

1. ALKALINE PHOSPHATASE

2. HORSERADISH PEROXIDASE