實驗概要
We provide a guideline procedure and tips for staining of paraffin embedded sections, including antigen retrieval, chromogenic detection and fluorescent detection.
The following protocol assumes the laboratory does not have an automated stainer or other capillary gap system that allows rapid application and rinsing of reagents (e.g. Shandon Sequenza). Reagents can be applied manually by pipet or the sequence of the protocol can be adapted to automated and semi-automated systems if these are available.
All incubations should be carried out in a humidified chamber to avoid drying of the tissue. Drying at any stage will lead to non-specific binding and ultimately high background staining. A shallow, plastic box with a sealed lid and wet tissue paper in the bottom is an adequate chamber, just as long as the slides are kept off the paper and can lay flat so that the reagents don’t drain off! A good solution is to cut a plastic serological pipette into lengths to fit your incubation chamber. Glue them in pairs to the bottom of the chamber, with the 2 individual pipette tubes of each pair being placed about 4.0 cm apart. This provides a level and raised surface for the slides to rest on away from the wet tissue paper.
Dilutions of the primary and secondary antibody are listed on the datasheets or are determined by testing a range. Adjust dilutions appropriately from the results obtained. Adhere strictly to all incubation times in the protocol.
For enzymatic methods, horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most commonly used enzymes. There are a number of chromogens used with these enzymes (see note x).
實驗步驟
Please refer to Notes section for the theory and section G for buffer recipes. If necessary, perform antigen retrieval before commencing with the following protocols.
Day 1
1. (If using an HRP conjugate for detection, blocking of endogenous peroxidase can be performed here but we recommend waiting until after the primary antibody incubation. See Day 2, step 2 and note v).
2. Wash the slides 2 x 5 minutes in TBS plus 0.025% Triton X-100 with gentle agitation. (See note i).
3. Block in 10% normal serum with 1% BSA in TBS for 2 hours at room temperature (See note iii).
4. Drain slides for a few seconds (do not rinse) and wipe around the sections with tissue paper (See note iv).
5. Apply primary antibody diluted in TBS with 1% BSA (See note v).
6. Incubate overnight at 4°C (See note iv).
Day 2
1. Rinse 2 x 5min TBS 0.025% Triton with gentle agitation.
2. If using an HRP conjugate for detection, incubate the slides in 0.3% H2O2 in TBS for 15 min (See note v).
3. For enzymatic detection (HRP or AP secondary conjugates):
Apply enzyme-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature.
For fluorescent detection:
Apply fluorophore-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature.
This step should be done in the dark to avoid photobleaching. |
4. Rinse 3 x 5min TBS.
If using fluorescent detection, end at this step and coverslip with mounting medium. If visualizing the protein with a chromogen, continue with the following steps.
5. Develop with chromogen for 10 min at room temperature (See note vi).
6. Rinse in running tap water for 5 min.
7. Counterstain (if required) (See note vii).
8. Dehydrate, clear and mount (See notes ix and x).
注意事項
1. The use of 0.025% Triton X-100 in the TBS helps to reduce surface tension, allowing reagents to cover the whole tissue section with ease. It is also believed to dissolve Fc receptors, therefore reducing non-specific binding. Abcam recommends TBS to give a cleaner background than PBS.
2. The secondary antibody may cross react with endogenous immunoglobulins in the tissue. This is minimized by pre-treating the tissue with normal serum from the species in which the secondary was raised. The use of normal serum before the application of the primary also eliminates Fc receptor binding of both the primary and secondary antibody. BSA is included to reduce non-specific binding caused by hydrophobic interactions.
3. The primary antibody should be diluted to the manufacturer’s recommendations or to a previously optimized dilution. If there is no suggested starting point, we recommend following the recommendations in Section A. Most antibodies will be used in IHC-P at a concentration between 0.5 and 10 μg/ml. Make sure the primary antibody is raised in a species different from the tissue being stained. If, for example, you had mouse tissue and your primary antibody was raised in a mouse, an anti-mouse IgG secondary antibody would bind to all the endogenous IgG in the mouse tissue, leading to high background. Use of mouse monoclonals on mouse tissue is discussed in our mouse-on-mouse protocol.
4. Overnight incubation allows antibodies of lower titer or affinity to be used by simply allowing more time for the antibodies to bind. Also, regardless of the antibody’s titer or affinity for its target, once the tissue has reached saturation point no more binding can take place. Overnight incubation ensures that this occurs.
5. H2O2 suppresses endogenous peroxidase activity and therefore reduces background staining. To check for the presence of endogenous peroxidases, incubate a tissue slide after re-hydration in a solution of DAB. If areas of the section appear brown under the microscope, a blocking step should help reduce this staining. Some epitopes are modified by peroxide, leading to reduced antibody:antigen binding. Incubating sections with peroxide after the primary incubation avoids this problem. Peroxide can be diluted in TBS or water. Some laboratories use methanol which is useful for blood smears or other peroxidase-rich tissues; peroxide diluted in methanol tends to reduce damage to the tissue caused by the reaction in aqueous solutions. For other tissue though, we recommend diluting in TBS or water. Reduced binding of some antibody:antigen pairs, in particular cell surface proteins, has been observed after methanol/peroxide incubation. (If using AP or fluorescent detection, omit peroxidase quenching as it only applies to HRP conjugates).
6. Develop the colored product of the enzyme with the appropriate chromogen. The choice depends on which enzyme label you are using, the colored end product you prefer and whether you are using aqueous or organic mounting media (See note xii for further details). Some commonly used substrates are listed below:
7. Some commonly used counterstains are hematoxylin (blue), nuclear fast red, or methyl green. When using fluorescent detection, DAPI (blue) or propidium iodide/PI (red) can be used to counterstain.
8. Don’t forget that DAB is a suspected carcinogen. Wear the appropriate protective clothing. Deactivate it with chloros in a sealed container overnight (it produces noxious fumes when chloros is added) and dispose of it according to laboratory guidelines. If using AP, add 0.24 mg/ml Levamisole (Sigma L9756) to the chromogen solution. Levamisole suppresses endogenous phosphatase activity and therefore reduces background staining.
9. If using AEC, Fast Red, INT or any other aqueous chromogen then don’t forget that they are alcohol soluble. Use a suitable aqueous mounting media. Don’t dehydrate and clear!
10. Dehydrate and clear DAB, New Fuchsin, Vega Red, NBT, TNBT or any other organic chromogen developed sections by sending them through the rehydration protocol listed in section C in the opposite order. Mount sections in a suitable organic mounting media. Sections mounted in organic mounting media have a better refractive index than those mounted in aqueous mounting media. This means that the image seen down the microscope will be sharper and clearer if organic mounting media is used.