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Experimental Methods for LSL series products ("#LSL-LB-" products)

Products mentioned above are antiserum that can be used in either freeze slicing or paraffin embedded slicing method. Read the following procedures before proceeding with the experiment.

 I. Freeze slicing method

  1. Freezing the tissue sample:
    While the tissue is still fresh, cut into suitable pieces and rapidly freeze them ie. with liquid nitrogen. For very small samples, use an specific agent designed for embedding and freezing. Wrap in aluminum foil before freezing. *1

  2. Creating thin sections:
    Make thin sections using a cryostat (~ - 20℃) and transfer them to glass slides. Then place your finger tip under the slide and warm the sample. Be sure that the sample sticks to the glass. Take extra caution to keep the tissue sample intact.

  3. Fixing the sample:
    Fix the sample with cold acetone or ethanol (less than –4℃) for 5 to 15 minutes. Avoid using formaldehyde. Evaporate the acetone or ethanol at room temperature for 1 to 2 minutes. Then wash the sample with PBS.

  4. Pre-treating the sample:
    The extracellular matrix, including collagen, becomes less immunoactive with aging. Adding protein or sugars into the sample may sometimes help improve the reaction. Refer to II-2 for details. Before proceeding to the immuno-staining procedure, treat the sample with a 2-5% of BSA and/or casein to prevent non-specific reactions. *2

 II. Paraffin embedded slicing method

When the sample is fixed with 4% para-formaldehyde and embedded in paraffin, clear stain pictures may not always be obtained using standard procedures. If this problem occurs, the result may be improved by the following enzyme treatments described below.

  1. Washing the sample:
    Remove paraffin from the sample and wash with PBS.

  2. Enzyme treatment:
    Add a few drops of either 0.1% pepsin (0.5M acetic acid), 0.4% pepsin (0.01N HCl), 1% trypsin (PBS), or hyaluronidase 25 mg/ml in PBS to the sample and keep at room temperature for 20 to 60 minutes. *3

  3. Blocking:
    After washing in PBS, in order to prevent non-specific reaction, add a 2-5% of casein and/or BSA. Then proceed to immuno-staining steps. *2


*1 Storing tissue clumps below –80℃ will preserve antigenic activity. However, repeated freeze/thawing, storing at temperatures above –40℃ or drying before fixing can significantly reduce the reactivity with antibodies. After the prep

*2 Use 1 to 3 % of casein, BSA and/or gelatin for blocking. Do not use gelatin for staining collagen.

*3 Concentration of enzymes, reaction times and temperatures are only an estimate. Take extra caution since an extended enzyme treatment may cause tissue degradation. Also when staining Type II collagen, treatment with hialuronidase will improve the result.

 III. General precautions regarding immunostaining

When staining the samples, it is important to consider the first and second antibody titers as well as reaction time and temperature after reconfirmation of the specificity of the antibodies. In order to improve the staining results, predetermine the optimal antibody dilution, reaction temperature and time by conducting a preliminary experiment.
The specificity of the LSL series products listed above, have been verified previously. However, the titer of the antibody differs depending upon the conditions of the reaction. Therefore, determine the optimum antibody dilution using the value given in the chart as the midpoint, also testing values above and below of this value. Repeat these steps for the second antibody. It is generally recommended to use a high dilution at low temperatures and long reaction time for clear pictures. It is also important to prepare control samples using the general staining method (ie. HE staining), to compare with pictures obtained from immunostaining.
References
Sasano Y, et al., BMPs induce direct bone formation in ectopic sites independent of the endochondral ossification in vivo. Anat Rec. 1993 Jun;236(2):373-80. PMID: 8338240
Mizoguchi I, et al., An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate. Histochemistry. 1990;93(6):593-9. PMID: 2184152
Horton WA, et al., Immunohistochemistry of types I and II collagen in undecalcified skeletal tissues. J Histochem Cytochem. 1983 Mar;31(3):417-25. PMID: 6338106

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.