Preparation of Tissue Fixation Embedding and Sectioning

1. Phosphate-buffered saline (PBS).

2. 4% Paraformaldehyde is prepared on the day of use by adding 4 g paraformaldehyde (BDH/Merk Ltd, Poole, UK)/100 mL PBS, heating at 65°C until the paraformaldehyde has dissolved and cooling on ice (aliquots can frozen but only freeze-thaw once). Paraformaldehyde is toxic, handle solutions within a fume cupboard.

Situ Hybridization Tissues

Fig. 1. Nonradioactive in situ hybridization on wax-embedded tissues in combination with immunohistochemical staining. Blue staining represents in situ hybridization signal and red is the reaction following immunohistochemical detection of other antigens. (A) In situ hybridization for cSox21 (a transcription factor, [8]) plus immunohostochemical staining for neurofilament (antineurofilament monoclonal (Dako Ltd). (B) In situ hybridization for cSox2 (9) plus immunohistochemical detection of BrdU to identify proliferating cells (see ref. 10 for further details). (C) In situ hybridization for cSox3 (8) plus immunohistochemical detection of Brachyury (a nuclear transcription factor; 11). (See color plate 5 appearing after p. 368.)

3. Saline (0.83% sodium chloride).

5. Histolene (Cellpath plc, Hemel Hempstead, UK). Less toxic alternative to xylene.

6. Paraffin wax, Tissue Tek pastilated.

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