PCNA antigen retrieval
Introduction
This protocol uses heat-induced epitope retrieval (HIER) prior to immunohistochemistry (IHC) to improve staining of the PCNA antibody (Sigma P8825). Without HIER, the PCNA antibody does not work using standard IHC protocols likely due to excessive cross-linking of the protein due to aldehyde fixation. A brief (5–15 min) heat treatment on sectioned tissue is sfficient to expose the PCNA antigen site. Although this protocol works well to expose the PCNA antigen site, prolonged heat exposure may destroy antigen sites of other antibodies. If immunolabeling using multiple antibodies is desired it must be empirically tested to determine if each antibody works after heat treatment. Antibody staining using GFP works well after heat treatment, but the intensity of the label is often weaker. For example, using GFP antibody after heat treatment in the Tg(gfap:EGFP) background usually results in only the soma of radial glial cells labelled in the retina. Without heat treatment, both the processes and soma are brightly labelled. Generally, a shorter heat treatment can improve labelling. Lastly, heat treatment will quench any uorescence. This approach cannot be used in conjunction with endogenous uorescence or after any uorescent antibodies have been applied to the tissue.
Protocol
Day 1
Buffers
10 mM Sodium citrate buffer, 0.5% Tween20 (pH 6) [1 L]
1X PBS, 0.1% Tween20 (PBT) [50 mL]
Blocking solution (1X PBS, 0.2% TritonX/Tween20, 2% normal goat serum ) [1 mL]
This protocol uses heat-induced epitope retrieval (HIER) prior to immunohistochemistry (IHC) to improve staining of the PCNA antibody (Sigma P8825). Without HIER, the PCNA antibody does not work using standard IHC protocols likely due to excessive cross-linking of the protein due to aldehyde fixation. A brief (5–15 min) heat treatment on sectioned tissue is sfficient to expose the PCNA antigen site. Although this protocol works well to expose the PCNA antigen site, prolonged heat exposure may destroy antigen sites of other antibodies. If immunolabeling using multiple antibodies is desired it must be empirically tested to determine if each antibody works after heat treatment. Antibody staining using GFP works well after heat treatment, but the intensity of the label is often weaker. For example, using GFP antibody after heat treatment in the Tg(gfap:EGFP) background usually results in only the soma of radial glial cells labelled in the retina. Without heat treatment, both the processes and soma are brightly labelled. Generally, a shorter heat treatment can improve labelling. Lastly, heat treatment will quench any uorescence. This approach cannot be used in conjunction with endogenous uorescence or after any uorescent antibodies have been applied to the tissue.
Protocol
Day 1
- Allow sections to dry for 2 hr at RT
- Pre-heat 10 mM sodium citrate, 0.5% Tween20 solution (pH 6) in coplin jar (with lid on to prevent overflow) by microwaving (until it’s about to boil).
- Place coplin jar in hybridization oven set to 95 C and allow it to equilibrate for 10 min
- Place slides in coplin jar and incubate at 95 C for 15 min, then immediately place slides in new coplin jar filled with PBT and let cool 10 min.
- Working quickly, take slides out of coplin jar, dry perimeter with kim wipe and draw boundary with PAP pen. Do not allow slides to dry out.
- Wash 2x 300 uL PBT, 5 min RT
- Apply 300 uL blocking solution, 1–2 hr RT
- Apply mouse anti-PCNA (1:1000, Sigma P8825) in blocking solution and incubate at 4 C O/N in humid chamber.
- Remove 1 antibody (can be reused 2x) and 4x wash in 300 uL PBT, 10 min RT
- Apply mouse anti-Cy3 (1:500) in blocking solution, 2–4 hr RT or 1–2 hr 37 C
- 4x wash in 300 uL PBT, 10 min RT
- Hoechst (10 mg/mL) in ddH2O, 15 min RT
- 2x wash 300 uL PBS, 5 min RT
- Apply 40 uL 80% glycerol, place coverslip on and seal with nail polish.
Buffers
10 mM Sodium citrate buffer, 0.5% Tween20 (pH 6) [1 L]
- 2.94 g tri-sodium citrate (dihydrate)
- 5 mL Tween20
- 950 mL ddH2O
- Adjust to pH 6 with 1 M HCl then top up to 1 L. Can be stored for 3 months at RT.
1X PBS, 0.1% Tween20 (PBT) [50 mL]
- 5 ml 10X PBS
- 50 uL Tween20
- 45 mL ddH2O
Blocking solution (1X PBS, 0.2% TritonX/Tween20, 2% normal goat serum ) [1 mL]
- 977 uL 1X PBS
- 2 uL TritonX-100 (for day 1) or Tween20 (for day 2)
- 20 uL normal goat serum