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    All-in-One Kit for Immunohistochemical Staining for Tissues with Antibodies

    互联网

    910

    实验试剂

     

    BioModuleTM

    PBS

    Ethanol [ 详细信息 : 95%-100%]

    Xylene

    DAB

     

    实验设备

     

    Light Microscope

    实验材料

     

    Coplin jars or staining containers

    frozen and formalin--xed paraffin embedded tissues

     

    实验步骤

     

    1. Obtain or prepare the formalin-fixed paraffin embedded sections of choice.

    Dry slides containing 4 µm formalin-fixed paraffin embedded sections in a 55ºC oven for 2 hours or overnight (do not allow the temperature to exceed 60ºC). Store the slides containing the formalin-fixed paraffin embedded tissue sections at room temperature until needed.

    1)   Place slides in xylene for 5 minutes at room temperature.

    2)   Remove slides and place in xylene a second time for an additional 5 minutes.

    3)   Remove slides and place in 100% ethanol 2 times for 5 minutes each time.

    4)   Remove slides and place in 95% ethanol for 5 minutes.

    5)   Remove slides and place in 80% ethanol for 5 minutes.

    6)   Remove slides and place in PBS for 10 minutes.

    7)   Drain any excess reagent by tapping the edge of the slide on paper towels and wipe the area near the tissue sections with a laboratory wipe.

    Circle each tissue section using the Mini PAP Pen.

    2. Proceed immediately to Peroxidase Quenching.

    Peroxidase Quenching

    Peroxo-Block™ works efficiently on many tissues, especially with formalin-fixed paraffin embedded tissues, but with some frozen tissues, you may need to use 3% H2O2 to obtain optimal results (better morphology or lower background). To use 3% H2O2 for blocking endogenous peroxidase activity, incubate the slides in 3% H2O2 in methanol for 10 minutes. Wash slides 3 times with 1X PBS for 2 minutes, each time and then proceed to primary antibody incubation.

    3. Epitope Retrieval Protocol

    A sample epitope retrieval protocol developed for some Zymed® Antibodies is described below. The epitope retrieval protocol is used to reverse the loss of antigenicity that occurs with some epitopes in formalin-fixed paraffin embedded tissues. Some Zymed® antibodies may require epitope retrieval protocol (e.g., estrogen receptor, and Ki-67 antibodies), while the staining of many other antibodies may be enhanced by epitope retrieval protocol (e.g, S-100, cytokeratin, and synaptophysin antibodies).

    You may use any other epitope retrieval protocol recommended by the supplier of your primary antibody.

    1)    Heat Induced Epitope Retrieval Protocol

    2)    Based on the reagent that you are using, dilute the reagent to 1X as follows:

    To 25 ml 20×Citrate Buffer, pH 6.0 solution, add 475 ml distilled water to obtain a 1X Citrate Buffer, pH 6.0 solution.

    To 25 ml 20X EDTA solution, pH 8.0, add 475 ml distilled water to obtain a 1X EDTA solution, pH 8.0.

    3 ） Place the slides in a slide rack and place the rack in a 1 L glass beaker containing 500 ml diluted citrate buffer (1X), pH 6.0 or diluted EDTA (1X) solution, pH 8.0.

    4 ） Place the beaker with slides on a hot plate. Heat the solution until it boils and continue boiling the solution for 15 minutes.

    5 ） Remove beaker from the hot plate and allow the contents to cool for 25 minutes at room temperature.

    6 ） Rinse slides with 1X PBS and proceed immediately to Incubation with Primary Antibody. 

    Epitope Retrieval Protocol Using Enzymatic Digestion

    Brief protocol using Digest-All™ Kit is described below. The Digest-All™ is a flexible and standardized system for tissue digestion and contains trypsin, ficin, and pepsin proteolytic enzymes. The degree of digestion is based on a standardized 10 minute incubation at 37°C. The trypsin solution is supplied with diluent so that different trypsin concentrations can be made to generate a range of digestion activity between that of Ficin and Pepsin. 

    1)   Perform this epitope retrieval protocol after Peroxidase Quenching.

    2)   Add the digestion enzyme of choice to your tissue sections  Ficin and pepsin may be applied directly from the bottle. Trypsin requires dilution: 1 drop Trypsin concentrate to 3 drops Trypsin Diluent. Mix well and apply to tissue sections.

    3)   Incubate for 10 minutes at 37°C.

    4)   Wash slides in several changes of 1X PBS and proceed immediately to Incubation with Primary Antibody.

    4. Incubation with Primary Antibody

    For best results, primary antibody dilution and incubation times need to be determined empirically and are dependent on sample preparation, antibody affinity, amount of antigen present, and antigen accessibility. Refer to the Primary Antibody section on page 10 for more details. The HRP Polymer Conjugate will react with mouse, rabbit, guinea pig, and rat primary antibodies.

    1)   Dilute primary antibody in Antibody Diluent as recommended by the antibody manufacturer or as determined by a titration experiment. Alternatively, you may use prediluted 2nd Gen primary antibodies.

    2)   Apply 100 µl or enough of the diluted primary antibody to completely cover the tissue on each section.

    3)   Incubate the slides in a humidified chamber for 30 minutes.

    4)   Wash slides 3 times with 1X PBS containing 0.05% Tween-20 for 2 minutes, each time.

    5)   Proceed immediately to Incubation with HRP Polymer Conjugate, below

    6 Incubation with HRP Polymer Conjugate

    1)   Apply 2 drops (100 µl) or enough of the ready-to-use HRP Polymer Conjugate (Reagent A)   to completely cover the tissue on each section.

    2)   Incubate the slides in a humidified chamber for 10 minutes.

    3)   Wash slides 3 times with 1X PBS containing 0.05% Tween-20 for 2 minutes, each time.

    4)   Proceed immediately to Signal Development, below.

    Signal Development

    1)   Dilute the 20X DAB Chromogen supplied with the kit in a sterile microcentrifuge tube just prior to use as follows:

    Distilled water                    1 ml

    Reagent B1                        1 drop

    Reagent B2                        1 drop

    Reagent B3                       1 drop

    Mix well. Protect from light and use within 1 hour.

    2)   Apply 2 drops (100 µl) or enough of the diluted DAB chromogen (prepared in Step 1) to completely cover the tissue on each section.

    Caution: DAB is a known carcinogen; handle with care.

    Incubate the slides in a humidified chamber for 5 minutes or until the desired signal intensity is reached.

    Incubation time can vary depending on the antibody.

    Rinse thoroughly with distilled water.

    Proceed immediately to Counterstaining, below.

    7 Counterstaining

    1)   Apply 2 drops (100 µl) or enough of the Hematoxylin Counterstain Reagent to completely cover the tissue on each section.

    2)   Incubate the slides in a humidified chamber for 1-3 minutes.

    3)   Rinse thoroughly with distilled water.

    4)   Dip the slide in ammonia water 1X PBS or (0.25% NH3) to blue the nuclei.

    5)   Rinse thoroughly with distilled water.

    6)   Dehydrate through increasing concentrations of ethanol (95%,100%) for 10-20 dips each. Clear in 2 changes of xylene or xylene substitutes for 10-20 dips each.

    7)   Proceed immediately to Mounting, below.

    8 Mounting

    1)   Apply 2-4 drops (100-200 µl) of Histomount™ mounting medium to the slide.

    2)   Apply a cover slip on the slide.

    3)   Allow the medium to dry at room temperature overnight.

    9 Microscopy

    Evaluate the results by examining the slides using a light microscope at 20x magnification. Interpret the results as described below.

     

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