SEM Pathology
Table of contents
1. Examination of common tissue samples
Staining properties of platinum blue
Staining properties of osmium tetroxide
2. Examination of renal biopsy samples
3. Advantages of LVSEMs and future tasks
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2. Examination of renal biopsy specimens #100001
Sample preparation and staining
Tabletop LVSEMs allow for the observation of “paraffin sections for light microscopy” that are prepared for the pathological diagnosis of renal biopsy specimens.4),5),6),7),8) Not only freshly sliced specimens but also previously prepared specimens can even be used to examine under LVSEM, once coverslips and mounting agents are removed. In certain cases, these old tissue sections can be visualized with excellent resolution after they are stained with a heavy metal. Here, we focus on glomerular lesions in the glomerulonephritis cases we have examined to date. We describe how to perform a “rapid three-dimensional analysis of renal biopsy sections,” key points of diagnosis, and our observations regarding the basic structure of glomeruli.
Sample preparation method
Sample preparation and observation
Renal tissue specimens obtained by biopsy or surgery are first treated with a commonly used fixative (such as 10 – 20% buffered formalin), dehydrated, and embedded in paraffin. These paraffin blocks are then cut into thin sections (5 – 10 μm in thickness), mounted on standard-size glass slides, and pretreated as described below.
| Deparaffinization | 100% xylene (3 times) → 100 – 50% ethanol → Distilled water (When examining samples previously used for light microscopy, their coverslips need to be removed prior to proceeding to the steps utilizing 100 – 50% ethanol and distilled water. This can be achieved by completely immersing them in an organic solvent, such as xylene.) |
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| Staining | Select an appropriate stain solution based on research objectives.
(a) Platinum-blue staining: adjust the pH of the staining solution included in the TI-Blue kit (Nisshin EM Co., Ltd.) to 9, apply one drop of the solution to each tissue section, and incubate at room temperature for 10–20 min.
(b) Periodic acid-methenamine-silver (PAM) staining: Use the standard protocol, however HE staining can be omitted. (If tissue sections are already stained with HE, use them as they are.) |
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| Washing | Immerse samples in distilled water for 1 – 2 min. (Absorb excess water around a tissue section using, for example, a filter paper. Do not use a coverslip.)
Apply double-adhesive carbon tape to the center of an SEM sample stub and firmly attach a glass slide to the tape, positioning the tissue section to the center of the stub. |
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| Observation with a LVSEM | Accelerating voltage, 15 kV; vacuum, 30 – 50 Pa; backscattered-electron images |
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Staining
The heavy-metal stains “platinum-blue” and “PAM” are essential for thoroughly examining renal biopsy paraffin sections using a tabletop LVSEM. This is similar to the examination of commonly used normal tissue specimens. With renal tissue, the specificities of these two stains are complementary. They react with different types of cells and tissue, producing images with almost reversed patterns in brightness. This facilitates the careful analysis of the three-dimensional structure of glomerular lesions. When necessary, LVSEM images of a sample stained with a heavy metal can be compared with images of the same sample acquired with a light or fluorescence microscope.9) Staining with platinum-blue or PAM is effective in examining renal biopsy tissue. Here, we present the staining properties of these two heavy-metal stains, and show how they affect image brightness and contrast differently. We also describe how to examine biopsy samples collected from patients with glomerulonephritis.
(a) Staining properties of platinum-blue
Platinum-blue staining is a very simple process, requiring only to immerse samples in the stain solution adjusted to pH 9 for 10 – 20 min. Platinum-blue achieves histologically specific staining of renal tissue specimens (as in commonly used tissue specimens). The specificity results from differences in the staining intensity of cellular and tissue components, and leads to improved identification of various types of cells and tissues under a LVSEM. This feature of platinum-blue is especially useful in analyzing the podocytes (epithelial cells) of glomeruli and their surface structures such as foot processes.
(b) Staining properties of periodic acid-methenamine-silver (PAM)
PAM staining was developed in 1953 by David Jones as a silver impregnation technique specialized for evaluating renal tissue specimens. It was later improved by Gonpachi Yajima.10) This staining method is essential for analyzing the glomerular basement membrane, and thus has been routinely used for the histopathological examination of renal biopsy specimens under light microscopy. Under a LVSEM, the morphology of silver-stained basement membranes can be clearly visualized without removing (digesting) the podocytes and vascular endothelial cells that overlie the basement membranes. This is because the backscattered-electron signal from the basement membranes can pass through these overlying cell layers. PAM staining is suitable for analyzing, in addition to the glomerular basement membrane, the three-dimensional structures of the mesangial region and the components of the extracellular matrix, such as collagen fibers.
When evaluating renal biopsy specimens, it is essential to carefully examine the morphology of glomerular basic structures, i.e., podocytes (epithelial cells), foot processes, vascular endothelial cells, basement membranes, mesangial cells, and the mesangial matrix. The method described here provides both a whole-sample image (at lower magnifications) and a detailed view of particular structures in each part (at higher magnifications). Here, we present LVSEM images from several renal biopsy cases that can be obtained during actual examinations, and describe our findings regarding the basic structure of the glomerulus.
References
10) Kidney biopsy - atlas and text. Guidelines for standardizing the pathological diagnosis of renal biopsy specimens. (in Japanese) Tokyo Igakusha. 2010:56-67.
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