Demonstration of alpha 1-antitrypsin by immunofluorescence on paraffin-embedded hepatic and pancreatic tissue.

Studies were performed in an attempt to improve current immunohistological techniques for the demonstration of alpha 1-antitrypsin (A1AT) in formalin-fixed paraffin-embedded tissues. The unwanted fluorescence (UF) commonly occurring in such procedures was found to be effectively eliminated by immunoadsorption of A1AT antisera with human serum lacking A1AT (Pi-null phenotype) coupled in solid phase to glutaraldehyde-activated aminohexyl-Sepharose 4B. Specificity of the antisera for A1AT was established by subsequent solid phase immunoadsorption against normal human serum bound to AH-Sepharose 4B. Using these techniques, immunoreactive A1AT was demonstrated in the cytoplasm of hepatocytes in liver biopsies obtained from patients with Z and MZ serum phenotypes, and in the cytoplasm of normal pancreatic islet cells.     

Combined in situ zymography, immunofluorescence, and staining of iron oxide particles in paraffin-embedded, zinc-fixed tissue sections

Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue

Background  

Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues.     

Adherence of digested tissue sections for fungal immunofluorescence

Specific immunofluorescence (IF) staining is helpful and often decisive in diagnosis of deep mycoses by examination of host tissue sections. Brightness of fluorescence may be significantly enhanced by prior digestion of tissues with trypsin, but the treatment tends to separate sections from slides. This is avoided by replacement of albumin in the process by an inexpensive commercial product, Elmer's Glue-All. The entire IF staining procedure for fungi in tissue sections is reviewed here, with emphasis on adhesion and digestion of sections.     

Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.     

Extraction of cells from paraffin-embedded tissue sections for single-cell DNA cytophotometry

A simple technique is presented for the isolation of cells from paraffin-embedded tissues for Feulgen DNA cytophotometric investigations. Tissue fragments from paraffin blocks were deparaffinized in xylene, rehydrated and refixed in a formalin solution and incubated in a solution of 0.5 pepsin in 0.25% hydrochloric acid. After filtration through a 70 micron mesh and centrifugation, the cells were smeared upon a glass slide. Comparison between the results obtained with freshly prepared imprints and with pepsin-extracted cells of the same tumor showed that the extraction technique does not influence the Feulgen reaction or the DNA distribution pattern. Investigations carried out on bladder and embryonal carcinomas have demonstrated that the method permits an analysis of histologically or histochemically identified tumor cells within individual tissue areas.