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Protocol 2

2. Immunohistochemistry using Anti-Ganglioside Antibodies


Tadashi Tai~Head, Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan


An indirect method using immuno- fluorescence coupled to second antibody is a relatively simple, rapid, and easy immuno-histochemical procedure. It is best suited for the study of membrane antigens in addition to intra- and extracellular antigens, and may be applied to frozen tissue sections, to cells in suspension, and to cells attached to glass slides or coverslips. This method may be applied using the fluorescence-activated cell sorter for analysis of cell populations and simultaneous detection of two different antigens (for instance one with FITC, another with rhodamine-conjugated antibodies).

Materials:

   

Equipment:

Cryostat; JUNG CM3000

Light microscope

Fluorescence microscope;

Moisture chamber

Animals:

Rats (Wistar, 80-day-old)

Reagents:

Diethyl Ether

Phosphate buffered saline (PBS 10mM sodium dihydrogen phosphate, 0.15 M sodium chloride, pH 7.4 )

Bovine serum albumin (BSA)

 

Acetone

Mouse MAb to ganglioside

Mouse MAb for control experiment

FITC-conjugated goat F(ab')2 fragment to mouse IgM ( chain-specific)

FITC-conjugated goat F(ab')2 fragment to mouse IgG ( chain-specific)

N2 liquid

Fluorescence mounting medium

Plastics

Non-fluorescent glass slide

Micro cover glass

   

 

 

Protocol:

Preparation of frozen sections. Small pieces of fresh samples should be placed in air-tight containers with a snap cap to avoid desiccation. The container is then quickly frozen in liquid nitrogen or an acetone dry ice bath and stored in a ultra-low freezer (-100C or -80C) until required. For sectioning, the samples are cut at 7m thickness with a cryostat microtome and thaw mounted on a glass slide (cleaned with ethanol). The mounted sections are air-dried for 2 hours. Following fixation, the slides are immunostained as follows:

1. Fix in cold acetone for 5 min. at -20C.

2. Remove the cold acetone by air drying.

3. Incubate with 5% BSA in PBS for 15 min. at room temperature.

4. After washing with 5% BSA in PBS twice, sections are incubated with the hybridoma supernatant containing antiganglioside mAb overnight at 4C. The time of incubation may range from 30 min. to overnight as determined by experience.

5. Wash with 1% BSA in PBS 4 times.

6. Incubate in FITC-conjugated goat anti-mouse IgM F(ab')2 or FITC-conjugated goat anti-mouse IgG F (ab')2 diluted at 1:1000 each with 1% BSA in PBS for 1 hr. at room temperature.

7. Wash with 1% BSA in PBS 5 times.

8. Apply drops of fluorescence mounting medium on section and mount micro cover glass. Observe the stained sections by fluorescence microscopy.

Notes: (1) Use of unfixed cells or tissue is not recommended for immuno-histochemistry since nonspecific staining can occur. In addition, if living cells in suspension are used, only the membrane antigens are exposed to the antibody and when reacting with the surface antigen, several events may occur. The nature of the fixative can greatly influence immunoreactivity of cells and tissues studied by mAbs. Best results were obtained with sections treated with acetone. Several other factors influence immunoreactivity of samples: dehydration, temperature, pH, and embedding.

(2) Caution is recommended during the interpretation and validation of the results. One of the best controls in an immunohistochemical study is incubation nof the sample with the MAb preabsorbed with the purified antigen. In this case, a lack of immunostaining should be obtained. It is also recommended to use samples containing and lacking the antigen of interest. The positive and negative controls will indicate if the technique was performed properly.

(3) Some caution must be used in interpreting ganglioside localization based on immunohistochemistry, since a lack of immunorecognition of a ganglioside epitope on cells and tissues does not necessarily mean that ganglioside is absent. There are indications that a number of factors are involved in influencing the reactivity of mAbs with specific cells and tissues.

References:

(1) Kotani, M., Kawashima, I., Ozawa, H., Terashima, T., and Tai, T. (1993) Differential distribution of major gangliosides in the rat nervous system detected by specific monoclonal antibodies. Glycobiology, 3: 137-146.

(2) Ozawa, H., Kotani, M., Kawashima, I., Numata, M., Ogawa, T., Terashima,T., and Tai, T.(1993) Generation of a monoclonal antibody specific for ganglioside GM4: evidence for GM4 expression on astrocytes in chicken cerebellum. J. Biochem.(Tokyo), 114:5-8

3) Kotani, M., Kawashima, I., Ozawa, H., Ogura, K., Ishizuka, I., Terashima, T., and Tai, T. (1994) Immunohistochemical localization of minor gangliosides in the rat nervous system. Glycobiology, 4: 855-865.

 


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