Protocol - Fixation

Figure 5.1 Diagrammatic explanation of standardization of IHC via AR and test battery to achieve a maximal retrieval level by an optimal protocol of AR. The intensity of IHC (axis y) is inversely correlated with the time of formalin fixation (axis x) as indicated by a reduced slope. Three arrows indicate a potential maximal retrieval level that may equalize the intensity of IHC to a comparable result for routinely processed, paraffin-embedded tissues with various time of fixation. Reproduced with permission from Shi et alHistotechnol. 1999 22 177-192.

Morgan JM. A protocol for preparing cell suspensions with formalin fixation and paraffin embedding which minimises the formation of cell aggregates. J Cell. Pathol. 2001 5 171-180. 

During the tissue fixation process, proteins are cross-linked, causing some epitopes to become undetectable by the staining protocols.10 HIAR reverses this effect, allowing these epitopes to be stained, and therefore has become increasingly important for many IHC staining protocols.19-22 However, the available automated IHC platforms vary in their ability to perform online HIAR. 

Armed with the common techniques of molecular biology and immu-nocytochemistry, an investigator is in a good position to apply in situ hybridization to EM for localization of nucleic acids at the ultrastructural level. McFadden (9) has a review on such use of in situ hybridization techniques. In the review, McFadden has included details of some of his laboratory protocols needed in in situ hybridization from fixation and labeling to probe labeling to the hybridization steps for localization of specific RNAs at the EM level. His protocol for hybridization is outlined below ... 

Plant Cells and Tissues Structure-Function Relationships. Methods for the Cytochemical/Histochemical Localization of Plant Cell/Tissue Chemicals. Methods in Light Microscope Radioautography. Some Fluorescence Microscopical Methods for Use with Algal, Fungal, and Plant Cells. Fluorescence Microscopy of Aniline Blue Stained Pistils. A Short Introduction to Immunocytochemistry and a Protocol for Immunovi-sualization of Proteins with Alkaline Phosphatase. The Fixation of Chemical Forms on Nitrocellulose Membranes. Dark-Field Microscopy and Its Application to Pollen Tube Culture. Computer-Assisted Microphotometry. Isolation and Characterization of... 

This protocol describes one of several useful procedures for constructing DNA content frequency histograms that yield information about cell-cycle phase distributions. The method uses ethanol-fixation to prepare cells for staining with PI. 

Brock R, Hamelers IHL, Jovin TM. Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. Cytometry 1999 35 353-362. 

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