PubMed PMID: 18285257. from tissue fixed in BE70 was of substantially higher quality and quantity than that was recovered from formalin-fixed tissue. Furthermore, the BE70 fixative showed excellent RNA and DNA integrity compared with that of NBF fixative based on real-time polymerase chain reaction analysis results. Immunohistochemical staining was comparable for the antigen tested. In conclusion, BE70 is usually a non-cross-linking fixative that is superior to NBF and 70% ethanol with reference to biomolecule recovery and quality from paraffin-embedded tissue. Additional studies to compare the histomorphologic and immunohistochemical overall performance and power in a clinical establishing are required. to a test was used to evaluate RNA integrity for each fixative condition. Assessment of DNA Quantity and Quality DNA was extracted from a 1-mm tissue core24 using the QIAamp DNA FFPE Tissue kit (Qiagen), according to the manufacturers instructions. Prior to the final elution step, 40 l CTCF of elution buffer was applied to the column and incubated at room heat for 2 min, followed by centrifugation. DNA yield was determined using a NanoDrop ND-1000 UV spectrophotometer. DNA quality assessed using a BioScore Screening and Amplification kit (Enzo Life Sciences, Farmingdale, NY).24 In addition, we also performed real-time PCR using the TaqMan? Gene Expression reagent (Applied Biosystems). Briefly, quantitative real-time PCR was performed with 1 g DNA assayed in a 20 l reaction volume. The reactions were incubated for 2 min at 50C, 10 min at 95C for initial denaturing, followed by 50 cycles of 95C for 15 sec and 60C for 1 min in the 7500 real-time PCR system (Applied Biosystems). We decided the Ct-value for the gene to assess DNA integrity in triplicates. The MannCWhitney test was used to evaluate DNA integrity Acetophenone for each fixative condition. Results Histomorphology Fixatives are applied to mediate the preservation of tissue; however in their application, they are generally combined with some form of impregnation to allow for microtomy and staining of microscopic sections. Most commonly fixatives are combined with paraffin impregnation (embedding), and frequently stained with hematoxylin and eosin (H&E) as contrasting brokers. Figure 1 demonstrates the histomorphologic features of a mouse glomerulus comparing NBF with BE70, as well Acetophenone as the sequential development of BE70 from a base fixative of 70% ethanol, by the addition of PBS, glacial acetic acid, and glycerol. Mouse tissues were Acetophenone chosen as a continuation of our Acetophenone prior studies in pre-analytic variables.2,21 Ethanol (70%) results in greater shrinkage with poorer cytologic features, compared with NBF. The introduction of PBS as a buffering answer offsets these changes, including less shrinkage and improved cytologic detail. Figure 2 demonstrates the histomorphology observed with H&E staining, comparing BE70 and NBF in a panel of mouse tissues, including liver, spleen muscle, brain, pancreas, colon, lung, and skin. Overall BE70 results in histomorphologic features much like 70% ethanol, and not dissimilar to NBF. Eosin staining is usually more intense when stained under the same conditions. Chromatin is more condensed with BE70 fixation, and nucleoli are more prominent. Red blood cells drop their hemoglobin and are ghost like after BE70 fixation, and eosinophils are not recognizable on H&E. Open in a separate window Physique 1. Histomorphological assessment of different fixatives on mouse kidney. Mouse kidney tissue section stained with hematoxylin and eosin (H&E) after buffered ethanol 70% (BE70) (A), neutral-buffered formalin (NBF) (B), 70% ethanol (E) (C), 70% ethanol + 0.5 PBS (EP) (D), 70% ethanol + 1% glycerol + 0.5 PBS (EGP) (E), or 70% ethanol + 0.5% glacial acetic acid + 0.5 PBS (EAP) (F) fixation. Acetophenone Level bar, 20 m. Abbreviation: PBS, phosphate-buffered saline. Open in a separate window Physique 2. Comparison between buffered ethanol 70% (BE70) and neutral-buffered formalin (NBF) fixation. Tissue sections were stained with hematoxylin and eosin (H&E) after BE70 or NBF fixation. H&E images of liver (A), pancreas (B), spleen (C), duodenum (D), heart (E), lung (F), brain (G), and skin (H). Scale bar, 50 m. Immunohistochemical Staining Immunohistochemistry is usually a critical element of modern histopathology. As such, we performed immunohistochemistry for Ki-67, AQP1, and CD31 on normal mouse tissue to demonstrate the retention of quality of.