Immunofluorescence General Protocol

Summary

The protocol provided general steps for immunofluorescence. Please refer to the APPLICATIONS section on the front page of product datasheet or product webpage to determine if this product is validated and approved for use on cultured cell lines (IF-IC), paraffin-embedded samples (IF-P), or frozen tissue sections (IF-F). Please see product datasheet or product webpage for appropriate antibody dilution and unmasking solution. This protocol is recommended for both unconjugated and fluorophore conjugated antibodies. 

Materials

1. 20X Phosphate Buffered Saline (PBS): (CST #9808)
2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814)
3. Blocking Buffer: (1X PBS/5% normal serum/0.3% Triton™ X-100) 
4. Antibody Dilution Buffer: (1X PBS/1% BSA/0.3% Triton™ X-100) 
5. Fluorochrome-conjugated Secondary Antibodies
6. Prolong® Gold Antifade Reagent (CST #9071), Prolong® Gold Antifade Reagent with DAPI (CST #8961). 

Recipes and preparation:

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 16% Formaldehyde use fresh and store opened vials at 4°C in dark. Dilute 1 in 4 in 1X PBS to make a 4% formaldehyde solution.
2. To prepare 10 ml blocking buffer, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100. 
3. To prepare 10 ml antibody dilution solution, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA (#9998), mix. 

Reagents specific to IF-P application:

1. Xylene.
2. Ethanol, anhydrous denatured, histological grade, 100% and 95%.
3. Antigen Unmasking:

• For Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L add 2.94 g (C₆H₅Na₃O₇•2H₂O) to 1 L dH₂O. Adjust pH to 6.0.
• For EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C₁₀H₁₄N₂O₈Na₂•2H₂O) to 1 L dH₂O. Adjust pH to 8.0.

Procedure

A. Specimen Preparation

I. Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips. 

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.

   NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each. 
4. Proceed with Immunostaining (Section B).

II. Paraffin Sections (IF-P)

NOTE: Do not allow slides to dry at any time during this process. 

1. Deparaffinization/Rehydration:
   1).Wash three times in xylene for 5 min each.
   2).Wash two times in 100% ethanol for 10 min each.
   3).Wash two times in 95% ethanol for 10 min each.
   4).Rinse sections two times in dH2O for 5 min each.
   
   
2. Antigen Unmasking: NOTE: Consult product datasheet or product webpage for specific recommendation for the unmasking solution.
   1). For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0, then maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.
   2). For EDTA: Bring slides to a boil in 1 mM EDTA pH 8.0 followed by 15 min at a sub-boiling temperature. No cooling is necessary.
   
   
3. Proceed with Immunostaining (Section B).

III. Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section B). 
2. For fresh, unfixed frozen tissue, fix immediately, as follows:
   1). Cover sections with 4% formaldehyde diluted in warm 1X PBS.
   2). Allow sections to fix for 15 min at room temperature.
   3). Rinse slides three times in PBS for 5 min each.
   4). Proceed with Immunostaining (Section B).
   
   

B. Immunostaining

NOTE:All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading. 

1. Block specimen in blocking buffer for 60 min.
2. While blocking, prepare primary antibody by diluting as indicated on datasheet in antibody dilution buffer.
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
   NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section B, Step 8.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Coverslip slides with Prolong® Gold Antifade Reagent (CST #9071) or Prolong® Gold Antifade Reagent with DAPI (CST #8961).
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

Stats

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

• 

  Sergey Arkhipov Wednesday, 18 May 2016 - 00:54 UTC

  Thank you very much for this protocol. It is good to show to beginners and to have by experienced microscopists, too.