免疫组化实验方法

肺腺癌免疫组化	肺鳞癌免疫组化	肺泡癌免疫组化	肺透明细胞癌免疫组化

本文共包括三部分：1、酶免疫组化实验；2、荧光免疫组化实验；3、实验问题分析

PART ONE

Enzymatic Protocol 1

Contents

• Overview
• Materials
• Sample Preparation
• Tissue Fixation and Mounting - Cryostat Sections
• Tissue Fixation and Mounting - Paraffin-embedded Sections
• Tissue Staining Protocol - Chromogenic
• Support Protocols
  • Gel Coating Solution
  • Gel-coated Slides
  • Antigen-retrieval Protocol

Overview
R&D Systems provides monoclonal, polyclonal and biotinylated antibodies for immunohistochemical use. The following protocol has been developed and optimized by R&D Systems' Immunohistochemical Laboratory. R&D Systems' antigen affinity-purified polyclonal and monoclonal antibodies have been used to stain frozen cells and tissues, as well as paraffin-embedded tissues. Immunohistochemistry protocols may require modification depending on the type of tissue used. Each investigator should determine the optimal conditions and working dilutions of antibodies. If using R&D Systems' primary antibodies, refer to the specific product insert to obtain an approximate working dilution. For all other reagents, follow the manufacturer's instructions. For Research Use Only.

Materials

Primary Antibodies
Unlabeled or biotinylated antigen affinity-purified polyclonal antibodies (R&D Systems' AF or BAF series) or selected unlabeled or biotinylated monoclonal antibodies (R&D Systems' MAB or BAM series)

Cell & Tissue Staining Kits - Enzymatic/Chromogenic

• Against Goat Primary Antibodies:
  • AP-BCIP/NBT (R&D Systems Catalog # CTS007)
  • HRP-DAB (R&D Systems Catalog # CTS008)
  • HRP-AEC (R&D Systems Catalog # CTS009)
• Against Mouse Primary Antibodies:
  • AP-BCIP/NBT (R&D Systems Catalog # CTS001)
  • HRP-DAB (R&D Systems Catalog # CTS002)
  • HRP-AEC (R&D Systems Catalog # CTS003)
• Against Rabbit Primary Antibodies:
  • AP-BCIP/NBT (R&D Systems Catalog # CTS004)
  • HRP-DAB (R&D Systems Catalog # CTS005)
  • HRP-AEC (R&D Systems Catalog # CTS006

Buffers and Additional Supplies

• Fixative: 85 mM Na2HPO4, 75 mM KH2P04, 4% formaldehyde (Sigma Catalog # P6148) and 14% (v/v) saturated picric acid (Sigma Catalog # 925-40), pH 6.9. Picric acid is optional.
• Sucrose Solution: 130 mM Na2HPO4, 30 mM KH2PO4, 10% (w/v) sucrose (Sigma Catalog # S7903), 0.01% sodium azide (Sigma Catalog # S2002) and 0.03% Bacitracin (Sigma Catalog # B-0125), pH 7.2
• PBS: 50 mM Na2HPO4 and 140 mM NaCl, pH 7.2
• Incubation Buffer: 1% bovine serum albumin (Sigma Catalog # A2153), 1% normal donkey serum (Sigma Catalog # D9663), 0.3% Triton X-100 (Sigma Catalog # T9284) and 0.01% sodium azide (Sigma Catalog # S2002) in PBS
• Aqueous Mounting Medium (R&D Systems Catalog # CTS011)
• DAB Enhancer (R&D Systems Catalog # CTS010)
• Antigen Retrieval Reagents (R&D Systems Catalog # CTS013, CTS014, CTS015 or CTS016)
  

Note: Equivalent chemicals and reagents may be substituted for those listed above.

Sample Preparation (Frozen Tissues)
The vast majority of immunohistochemical procedures employ a cell or tissue fixation step using formaldehyde or other cross-linking fixatives prior to incubation with primary antibody. Fixation is required to retain tissue morphology and prevent degradation of tissue antigens. Fixation may be performed either by immersing dissected pieces of tissue (e.g. human biopsies) into the fixative, or by vascular perfusion (e.g. laboratory animals such as mice, rats, guinea pigs, etc.). It is very important to optimize fixing conditions since under- or over-fixation may reduce or abolish tissue immunoreactivity. The easiest way to correct under-fixation is to post-fix tissue sections on the slide before starting immunohistochemical staining. To recover antigens in over-fixed tissues, either protease-induced epitope retrieval (PIER) or heat-induced epitope retrieval (HIER) techniques are recommended. HIER can be performed using a microwave oven, pressure cooker, vegetable steamer, autoclave or water bath. After tissues are fixed, they may either be embedded into paraffin or covered with OCT compound and frozen for further sectioning. Paraffin-embedded tissues are cut using a microtome at room temperature, whereas frozen tissues are cut using a cryostat at temperatures below 0?C. Antigen immunoreactivity was found to be better preserved in frozen rather than paraffin-embedded tissues.^1,2

References

1. Larsson, L.-I. (1988) Immunocytochemistry: Theory and Practice, CRC Press, Boca Raton, Florida.
2. Frost, A. et al. (2000) Methods of antigen recovery vary in their usefulness in unmasking specific antigens in immunohistochemistry, Appl. Immunohistochem. Mol. Morphol. 8:236.
   

Tissue Fixation and Mounting - Cryostat Sections

1. Fix the tissue by vascular perfusion with 500 - 700 mL of Fixative.
2. Perfuse the animal with 400 mL of Sucrose Solution.
3. Dissect the tissue, mount in OCT and freeze at -20 to -80?C.
4. Cut 5 - 15 mm thick tissue sections using a cryostat.
5. Thaw-mount the sections onto gel-coated slides. Refer to the Support Protocols section to follow for instructions on how to prepare gel-coated slides.
6. Dry the slides for 30 minutes on a slide warmer at 37?C. Slides containing cryostat sections can be stored at -20 to -70?C for up to 12 months.

Tissue Fixation and Mounting - Paraffin-embedded Sections

1. Fix the tissue by vascular perfusion with 500 - 700 mL of Fixative.
2. Dissect the tissue.
3. Immerse the tissue in 70% ethanol three times for 30 minutes each at room temperature.
4. Immerse the tissue in 90% ethanol two times for 30 minutes each at room temperature.
5. Immerse the tissue in 100% ethanol three times for 30 minutes each at room temperature.
6. Immerse the tissue in toluene three times for 20 minutes each at room temperature.
7. Embed the tissue in paraffin (Paraplast, Fisher Scientific) two times for 60 minutes each at 58?C. Alternatively, tissues can be embedded into paraffin using specialized automated tissue processing systems.
8. Cut 5 - 15 祄 thick tissue sections using a rotary microtome.
9. Float the sections in a 56?C water bath.
10. Mount the sections onto histological slides.
11. Dry the slides overnight at room temperature. Slides containing paraffin-embedded sections can be stored at room temperature.

When it is not possible to fix tissue by perfusion, dissected tissue may be fixed by immersing the tissue into a 10% formalin solution for 4 - 8 hours at room temperature. It is commonly accepted that the volume of fixative should be 50 times greater than the size of the immersed tissue. Avoid fixing the tissue for greater than 24 hours since tissue antigens may either be destroyed or masked (A.C. Cuello, ed., 1993, Immunohistochemistry: Methods in the Neurosciences, Vol. 14; IBRO Handbook Series, John Wiley & Sons, New York).

Tissue Staining Protocol - Chromogenic
This protocol is based on using R&D Systems' AP-BCIP/NBT Cell & Tissue Staining Kits. Refer to the protocol booklet provided with each kit for technical notes. Steps may vary depending on the chromogenic substrate. Refer to the Materials section of this protocol for additional substrates.

1. Prepare the slides as follows.
   
   • Cryostat tissue sections
     Thaw the slides for 10 minutes at room temperature.
   • Paraffin-embedded tissue sections
     1. Immerse the slides in Xylene (mixed isomers) two times for 10 minutes each.
     2. Immerse the slides in 100% alcohol (denatured) two times for 10 minutes each.
     3. Immerse the slides in 95% alcohol once for 5 minutes.
     4. Immerse the slides in 70% alcohol once for 5 minutes.
     5. Immerse the slides in 50% alcohol once for 5 minutes.
     6. Rinse the slides in deionized water.
2. Rehydrate the slides in PBS for 10 minutes.
3. Drain the slides and wipe off excess buffer.
4. Perform antigen-retrieval, if necessary. Refer to the Support Protocols section to follow.
5. Incubate the sample with 1 - 3 drops of Serum Blocking Reagent for 15 minutes.
6. Drain the slides and wipe off excess reagent. Do not rinse.
7. Incubate the sample with 1 - 3 drops of Avidin Blocking Reagent for 15 minutes.
8. Rinse the sample with PBS. Drain the slides and wipe off excess buffer.
9. Incubate the sample with 1 - 3 drops of Biotin Blocking Reagent for 15 minutes.
10. Rinse the sample with PBS. Drain the slides and wipe off excess buffer.
11. Incubate the sample with primary antibody diluted in Incubation Buffer. Follow the manufacturer's recommended working dilution, incubation time and incubation temperature.
    Note: An overnight incubation at 4?C is recommended when using primary antibodies from R&D Systems.
12. Rinse the sample with PBS and then wash 3 times in PBS for 5 minutes each. Drain the slides and wipe off excess buffer.
13. Incubate the sample with 1 - 3 drops of Secondary Biotinylated Antibodies for 30 - 60 minutes. Adjust the incubation time depending on the thickness of the tissue section.
14. Rinse the sample with PBS and then wash 3 times in PBS for 15 minutes each. Drain the slides and wipe off excess buffer.
15. Incubate the sample with 1 - 3 drops of HSS-AP for 30 minutes.
16. Rinse the sample with PBS and then wash 3 times in PBS for 2 minutes each.
17. Rinse the sample with distilled water. Drain the slides and wipe off excess water.
18. Incubate the sample with 1 - 5 drops of BCIP/NBT Chromogen for 20 - 30 minutes. Monitor the intensity of tissue staining under a microscope.
19. Rinse the sample with PBS and then wash in PBS for 10 minutes.
20. Rinse the sample with distilled water. Drain the slides and wipe off excess water.
21. Mount the sample either without counterstain or after counterstaining with Methyl Green or Nuclear Fast Red.
22. Drain the excess mounting medium by placing the slides vertically on filter paper or a paper towel. Allow the slide to dry.
23. Examine the slides under a microscope.
    

Support Protocols

Preparation of gel-coated slides

Gel Coating Solution:

1. Dissolve 5 g gel [type A, 175 bloom from porcine (Sigma Catalog # G2625)] in 1 L water, while heating. Do not allow the temperature to exceed 45?C.
2. Add 0.5 g chromium potassium sulfate [CrK(SO4)2 . 12H2O] and dissolve completely. Store at 4?C. This solution can be re-used 3 - 4 times and is stable for 1 - 2 months when stored at 4?C.

Gel-coated Slides:

1. Heat the Gel Coating Solution to 40 - 44?C.
2. Filter the solution using coarse filter paper.
3. Pour the solution into a large staining dish.
4. Remove bubbles from the surface of the solution.
5. Briefly submerge a rack containing histological slides into the solution.
6. Remove the rack of slides from the staining dish.
7. Place the rack onto a paper towel and cover the slides with paper towels to avoid contamination with dust.
8. Allow the slides to dry at room temperature. Dry slides can be stored for 1 year at -20?C.

Antigen-retrieval Protocol
This protocol is based on using R&D Systems' Antigen Retrieval Reagents (Catalog # CTS013, CTS014, CTS015 or CTS016). Note: the antigen-retrieval capacity of each Antigen Retrieval Reagent depends on sample preparation, antigen structure, incubation time (up to 30 minutes) and temperature (90 - 100?C). Each investigator should determine optimal conditions.

1. Dilute the 10X Antigen Retrieval Reagent 10-fold, using deionized water, to make the Retrieval Solution.
2. Heat the Retrieval Solution to 92 - 95?C. This may be accomplished by placing a polypropylene Coplin staining jar filled with Retrieval Solution into a water bath. Note: heating may crack glass staining dishes.
3. Insert the slides containing tissues into the heated Retrieval Solution and incubate 2 - 10 minutes. Note: cryostat sections are more susceptible to the damaging effects of the Retrieval Solution than paraffin-embedded tissues. To avoid tissue damage, it may be necessary to shorten the incubation time to 2 - 5 minutes for cryostat sections.
4. Place the Coplin jar containing the Retrieval Solution and slides on a lab bench and allow to cool for 5 - 10 minutes at room temperature.
5. Rinse the slides with distilled water followed by PBS. Note: tissues may become loose after the retrieval procedure, avoid vigorous rinsing to prevent detachment of the tissues from the slides.
   Proceed to step 5 of the Tissue Staining Protocol.
   

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