Paraffin sections

For light microscopic examination, liver tissue was fixed in 10 % buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. In some cases, preparations were stained with PTAH (phosphotungstic acid-haematoxylin), by the Van Gieson method and the PAS (periodic acid-Schiff)... 

TABLE 1.3 Comparison of pRB-IHC between Frozen and Paraffin Sections Using Four Protocols of AR... 

Shi SR, Cote RJ, Yang C, et al. Development of an optimal protocol for antigen retrieval a test battery approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections. J. Pathol. 1996 179 347-352. 

Ferrier CM, van Geloof WL, de Witte HH, et al. Epitopes of components of the plasminogen activation system are re-exposed in formalin-fixed paraffin sections by different retrieval techniques. J. Histochem. Cytochem. 1998 46 469-476. 

Elias JM, Margiotta M. Low temperature antigen restoration of steroid hormone receptor proteins in routine paraffin sections. I. Histotechnol. 1997 20 155-158. 

Nasr SH, Galgano SJ, Markowitz GS, et al. Immunofluorescence on pronase-digested paraffin sections a valuable salvage technique for renal biopsies. Kidney Int. 2006 70 2148-2151. 

Frank TS, Svoboda-Newman SM, Hsi ED. Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR. Diagn. Mol. Pathol. 1996 5 220-224. 

Taylor CR, Mason DY. The immunohistological detection of intracellular immunoglobulin in formalin-paraffin sections from multiple myeloma and related conditions using the immunoperoxidase technique. Clin. Exp. Immunol. 1974 18 417 29. 

Von Boguslawsky K. Immunohistochemical detection of progesterone receptors in paraffin sections. APMIS 1994 102 641-646. 

Huang S-N. Immunohistochemical demonstration of hepatitis B core and surface antigens in paraffin sections. Lab. Invest. 1975 33 88-95. 

Notes. Mouse tissues were fixed with 4% formaldehyde for 6h. Paraffin sections were boiled in 20 mM Tris-HCl buffers (TB), at pH 6.0 or at pH 9.0, for 10 min. After cooling, the sections were briefly washed with distilled water and heated in another buffer (e.g., pH 9.0 and then 6.0) for 5min. Some specimens treated in the second buffer were heated in the first buffer (e.g., pH 9.0, then pH 6.0, and finally pH 9.0 pH 9-6-9) for 5 min. The antigens were localized in respective tissues described in Table 17.1. Immunostaining was scored as follows +++, strong ++, moderate +, weak , faint -, negative. 

Shi SR, Chaiwun B, Young L, et al. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J. Histochem. Cytochem. 1993 41 1599-1604. 

Prior to immunohistochemical staining, paraffin sections must be properly mounted onto slides, and then deparaffinized and rehydrated. To help adherence to the glass and decrease the chances of sections dissociating from the slides, paraffin tissue sections should be mounted on tissue-adhesive-coated slides. The use of tissue-adhesive-coated slides is especially important for paraffin tissue sections undergoing heat-induced antigen retrieval (see Sect. 6.1.1). 

As for paraffin sections, it is advisable to mount cryosections also onto adhesive-coated slides in order to decrease the chances of sections dissociating from the slides in the course of immunohistochemical staining. Once mounted on slides, cryosections are air-dried and fixed, usually in acetone or methanol. Aldehyde... 

Staining procedure for paraffin sections using ABC system... 

Immunohistochemistiy Tumor samples were fixed in formalin for 24 hours at RT and then embedded in paraffin. Serial paraffin sections of 3-4 pm were cut, placed on superffost plus slides, dewaxed with xylene, hydrated from ethanol to tap water, and transferred to PBS. Endogenous peroxidase activity was blocked by incubating slices with 3% H202 in PBS for 10 . 

Histology. Whole mussels were fixed for 1-2 days in Helly s fluid (10) and stored in 70% ethanol. Tissue was blocked at 2 mm thickness, embedded in paraffin, sectioned at about 7 ym, stained with hematoxylin and eosin, and mounted on glass slides using standard procedures. 

Antigen Retrieval for Immunohistochemical Reactions in Routinely Processed Paraffin Sections... 

Since HIER protocols are very harsh on tissue, it is essential to apply an adhesive to the glass slide to allow a strong attachment of the tissue. Paraffin sections of... 

If epoxy resin-embedded tissue is used, cut 2- jm-thick sections with a glass knife, mount on APES-coated slides, and dry as described in Note 2. Deplasticize the sections by immersing them in sodium eth(meth)oxide for 15 min (8). Wash the sections twice with equal parts of methanol (or IMS) and xylene, twice with methanol, for 3 min each, and rehydrate. Afterward, the same HIER and immuno-histochemical protocols are employed as in paraffin sections. 

Huang, S. N., Minassian, H., and Moore, J. D. (1976) Application of immuno-fluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest. 35, 383-390. 

Krenacs, L., Harris, C. A., Raffeld, M., andJaffe, E. S. (1996) Immunohistochemi-cal diagnosis of T-cell lymphomas in paraffin sections. J. Cell. Pathol. 1,125-136. 

As previously mentioned, the principles of staining are identical to those used in paraffin sections. The techniques used may be direct or indirect as described in Chapters 15-18. Indirect techniques are generally more sensitive and, therefore, preferable. Indirect techniques can be broken down into three steps. In the first step, an antibody directed against the antigen of interest is applied to the tissue section. In the second step, a labeled secondary antibody directed against the first antibody is applied. The last step consists of a detection step that is composed of linking the secondary antibody to a detection system and... 

For the following protocol, use 5- im-thick paraffin or frozen sections. Paraffin sections and frozen sections should be mounted on charged or appropriately coated glass slides (see Notes 1 and 2) (see Chapters 9 and 12). 

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