Cutting sections of paraffin-embedded tissues

Cold Spring Harbor Protocols (Impact Factor: 4.63). 05/2008; 2008(6):pdb.prot4987. DOI: 10.1101/pdb.prot4987
Source: PubMed


INTRODUCTIONThis protocol describes the sectioning of tissues embedded in paraffin blocks. Paraffin sections require extensive fixation and processing steps but provide superior morphology compared with other sectioning methods. Sectioning paraffin blocks requires experience and should be learned from an experienced researcher, if possible.

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    • "Hematoxylin and eosin (HE) staining was performed on longitudinal and transverse sections of wildtype and ae3 null adult mouse heart, using previously described protocols [45,46]. Briefly, paraffin-embedded hearts were sectioned into 3 μm slices, which were trimmed and floated onto a water bath at 42°C, containing 50 mg/l of gelatin while gently stretching the cut sections to avoid wrinkles. "
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    ABSTRACT: Background: Cardiac hypertrophy is central to the etiology of heart failure. Understanding the molecular pathways promoting cardiac hypertrophy may identify new targets for therapeutic intervention. Sodium-proton exchanger (NHE1) activity and expression levels in the heart are elevated in many models of hypertrophy through protein kinase C (PKC)/MAPK/ERK/p90RSK pathway stimulation. Sustained NHE1 activity, however, requires an acid-loading pathway. Evidence suggests that the Cl-/HCO3- exchanger, AE3, provides this acid load. Here we explored the role of AE3 in the hypertrophic growth cascade of cardiomyocytes. Methods: AE3-deficient (ae3-/-) mice were compared to wildtype (WT) littermates to examine the role of AE3 protein in the development of cardiomyocyte hypertrophy. Mouse hearts were assessed by echocardiography. As well, responses of cultured cardiomyocytes to hypertrophic stimuli were measured. pH regulation capacity of ae3-/- and WT cardiomyocytes was assessed in cultured cells loaded with the pH-sensitive dye, BCECF-AM. Results: ae3-/- mice were indistinguishable from wild type (WT) mice in terms of cardiovascular performance. Stimulation of ae3-/- cardiomyocytes with hypertrophic agonists did not increase cardiac growth or reactivate the fetal gene program. ae3-/- mice are thus protected from pro-hypertrophic stimulation. Steady state intracellular pH (pHi) in ae3-/- cardiomyocytes was not significantly different from WT, but the rate of recovery of pHi from imposed alkalosis was significantly slower in ae3-/- cardiomyocytes. Conclusions: These data reveal the importance of AE3-mediated Cl-/HCO3- exchange in cardiovascular pH regulation and the development of cardiomyocyte hypertrophy. Pharmacological antagonism of AE3 is an attractive approach in the treatment of cardiac hypertrophy.
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    ABSTRACT: Background: The integration of histology and microcirculation in real time through specific regions of the brain is a challenging concept that has not been reported before. This study describes for first time a brain-mapping model that superimposes regional microvascular blood flow (RMBF) analysis with immunohistochemistry analysis in an experimental ovine model. Methods: Five Merino sheep were instrumented, ventilated and cardiovascularly supported according to local guidelines. Two ultrasound catheter sheaths were inserted into the right internal jugular vein for the introduction of an intracardiac echocardiography probe and transeptal catheter, as previously described. For the analysis of RMBF, color-coded microspheres were injected into the left atrium while a reference blood sample was extracted from the femoral artery. After euthanasia and fixation with formalin, the brain was used as proof of principle and the endpoint for determination of microcirculation and histology analysis at different time points. An antero-posterior slicing strategy of the sheep brain differentiated even-numbered from odd-numbered slices. For the histology analysis, immunohistochemistry applied to odd-numbered slices used amyloid precursor protein (APP) antibodies and hematoxylin-eosin staining. Simultaneously, even-numbered slices were dedicated for cytometric quantification of RMBF. Results: Homogeneous allocation of microspheres to different regions of the brain over time with no statistical difference between slices and RMBF count was found. In addition, immunohistochemistry showed baseline staining, confirming a state of normal cerebral perfusion. Conclusions: This study has demonstrated the feasibility and reproducibility of a brain-mapping model that superimposes RMBF data and immunohistochemistry data over time, establishing a new experimental model.
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