Immunofluorescence With Dual Microwave Retrieval of Paraffin-Embedded Sections in the Assessment of Human Renal Biopsy Specimens

Abstract

Immunofluorescence of frozen tissue sections (IF-F) is a classic technique for renal immunopathologic examination. However, it has certain disadvantages, such as diffuse antigen distribution and few or even no glomeruli in the section. We developed a new technique of immunofluorescence staining using dual microwave retrieval in paraffin-embedded renal tissue sections (IF-DMP) and compared IF-DMP with IF-F in 406 renal biopsy samples. IF-DMP detected significantly more glomeruli than did IF-F (P< .001). There was no significant difference for the specificity and sensitivity in the detection of immunoglobulins, complements, κ, and λ between IF-F and IF-DMP. Concordant observations were 98% for all immunofluorescence, complements, κ, and λ staining and 100% for immunoglobulin staining. Both techniques were completely accurate in confirming diagnoses of various glomerular diseases. IF-DMP provided clearer images of tissue structure and more precise localization of antigens, and it is a suitable alternative for traditional IF-F in clinical renal immunopathologic diagnosis.

Paraffin immunofluorescence for detection of immune complexes in renal biopsies: an efficient salvage technique for diagnosis of glomerulonephritis in dogs

“…In one study, three methods of AR (heating with a Tris buffer of unknown pH, heating with CB, and pronase digestion) were tested, and it was demonstrated that heating with Tris buffer was most effective, allowing the evaluation of ICs in renal biopsy specimens [ 9 ]. The efficacy of dual MW-heating methods using an EDTA buffer (pH 8.0) has also been previously reported [ 8 ]. In this study, MW heating using a routine protocol (5 min pre-warming, 10 min MW, and 20 min cooling) was tested using CB (pH 6.0) as a standard AR solution and TEB (pH 9.0) as a high pH AR solution.…”

“…IF on FFPE (IF-FFPE) sections has been explored as an alternative to standard IF-F. The sensitivity and specificity of this method for the detection of ICs could be improved by prior antigen retrieval (AR) using proteolysis [ 6 , 7 ], heat [ 8 ], or a combination of both [ 6 ]. However, the efficacy of IF-FFPE for the detection of ICs in dog renal biopsies has not been evaluated.…”

Paraffin Immunofluorescence: A Valuable Ancillary Technique in Renal Pathology

“…Multiple proteolytic enzymes have been used in IF-P, including trypsin, 7 , 8 , 9 , 10 , 11 , 12 pronase E (protease XIV), 13 , 14 , 15 , 16 , 17 proteinase XXIV, 18 and proteinase K. 13 , 19 , 20 , 21 , 22 Successful results were also obtained by heat treatment with Tris or citrate buffers 15 and with dual microwave heating in EDTA antigen-retrieval solution. 23 In our laboratory, we use the pronase technique, which was originally described by Fogazzi et al. 17 and was introduced to the practice of renal pathology in the United States by Professor Vivette D’Agati (Columbia University, New York, NY).…”

Microwave-Assisted Tissue Preparation for Rapid Fixation, Decalcification, Antigen Retrieval, Cryosectioning, and Immunostaining

“…The above protocol is applicable for staining of cryosections (see also [42]). See also immunofluorescence microscopy of paraffin-embedded samples by other authors [35, 37]. …”