Recommended Antigen Unmasking Procedures for F4/80 Staining of Paraffin-Embedded Tissues

F4/80 is a 160 kDa cell surface glycoprotein predominantly expressed on murine macrophages. Clone CI:A3-1 (MCA497) specifically recognizes the F4/80 antigen and has been used extensively to detect the F4/80 antigen in paraffin sections.

Like many antigenic determinants, the F4/80 antigen can be masked by prolonged formalin-fixation and the paraffin-embedding process. Clone CI:A3-1 has been shown to work successfully on formalin-fixed paraffin-embedded tissue without pre-treatment, but prolonged fixation does mask the antigen.

Unmasking procedures are recommended to achieve optimum staining. An extensive study has been undertaken by AbD Serotec to establish the best conditions required for tissues fixed over varying periods of time. The following guidelines should be used alongside Protocol 7 - Staining of Paraffin-Embedded Tissues.

1. Enzyme Pre-Treatment using Proteinase K (Recommended for Tissues Fixed for 24 hours in Neutral Buffered Formalin, NBF)

Reagents

A. TE buffer (50 mM Tris base, 1 mM EDTA, pH 8.0)

Tris Base, 6.10 g
EDTA, 0.37 g
Distilled water, 1000 ml
Mix to dissolve. Adjust pH to 8.0 using concentrated HCl (10 M HCl). Store at room temperature.

B. Proteinase K stock solution (20x, 400 μg/ml in TE buffer, pH 8.0)

Proteinase K, 4 mg
TE buffer, pH 8.0, (Reagent A) 10 ml
Mix well. Store in aliquots at -20°C.

C. Proteinase K working solution (1x, 20 μg/ml in TE buffer, pH 8.0)

Proteinase K stock solution (20x), (Reagent B) 1 ml
TE Buffer, pH 8.0, (Reagent A) 19 ml
Mix well. Discard working solution after use.

Method

1. Dewax paraffin sections and rehydrate using preferred procedure.
2. Cover sections completely with Proteinase K working solution and incubate for 3 minutes at room temperature.
3. Rinse sections with Phosphate Buffered Saline (PBS).
4. Proceed with serum blocking and preferred staining protocol.

2. Heat-mediated Antigen Retrieval using Citrate Buffer, pH 6.0 (Recommended for Tissues Fixed for 7 Days or More in Neutral Buffered Formalin, NBF)

Reagent

Citrate buffer (10 mM citric acid, pH 6.0)

Citric acid (anhydrous), 1.92 g
Distilled water, 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1 M NaOH (be sure to mix well). Store this solution at room temperature for 3 months, or at 4°C for longer usage.

Method

1. Dewax paraffin sections and rehydrate using preferred protocol.
2. Pre-heat sodium citrate buffer in a staining vessel to 95-100°C.
3. Immerse slides in the citrate buffer and incubate for 10 minutes at 95-100°C. Check the citrate buffer level, add more if necessary, and then incubate for a further 10 minutes at 95-100°C.
4. Allow sections to cool for 20 minutes.
5. Rinse sections with PBS.
6. Proceed with serum blocking and preferred staining protocol.

Recommended fixative for CI:A3-1

Neutral buffered formalin (NBF)
 

Recommended positive control tissue

Liver, lymph node