Preparation of paraffin blocks
Includes dehydration of the samples in successive alcohols of increasing concentration, rinsed in xylol and mounting in paraffin.
1 to 10 sample batch
Sectioning of paraffin blocks*
Section cutting of the sample embedded in paraffin with a thickness between 5 and 20 microns, floating the sections in a hot bath and mounting the sections on slides. We use albumin as an adhesive when the sections are going to be stained, if they are to be used for immunohistochemistry, special slides with a positive charge are used with an associated increased cost.
Hematoxylin and Eosin staining
Harris hematoxylin (Pap smear solution) and an alcoholic eosin solution are used for staining the sections; the sections are dehydrated and covered, using Entellan as a mounting medium.
Preparation of a frozen block
Impregnation of histological samples in 30% sucrose in phosphate-saline buffer, and subsequent coating with OCT for freezing in dry ice and storage at -70 ° C.
Cutting of frozen blocks*
The frozen sample is cut in a cryostat at a temperature of -20ºC (it can vary according to the hardness of the tissue), obtaining sections with a thickness of 10 microns or greater. The sections are floated in cold phosphate-saline buffer and mounted on the slides. When the sections are to be stained, albumin is used as an adhesive. When the slides will be used for immunohistochemistry, special slides with positive charge are used with an associated increased cost.
This includes the following successive treatments of the sections: unmasking of the antigen in a specific buffer at a high temperature, blocking with non-immune serum and triton X-100, incubation with the primary antibody and subsequent detection with a conjugated secondary antibody conjugated to a fluorochrome. Hoechst is included in the mounting medium used to cover the sections in order to visualize the nuclei of the cells present in the sections.
Immunoperoxidase with signal amplification*
This includes the following successive treatments of the sections: inhibition of endogenous peroxidase with hydrogen peroxide, unmasking of the antigen with a specific buffer at a high temperature, blocking with non-immune serum and triton X-100 and incubation with the primary antibody. A signal amplification technique is used for the detection of the antibody, which consists of successive incubations with a secondary antibody conjugated to biotin, and also a solution of streptoavidin conjugated to peroxidase. Finally, the samples are treated with hydrogen peroxide and diaminobenzidine which reacts with the peroxidase to produce a brown product. The cell nuclei are be stained gently with Harris hematoxylin.
* If the sections cause problems due to the consistency of the fabric or a problem in the preparation of the block, the cost will be higher, depending on the additional working time
* The primary antibody must be supplied by the client.