Modified Silver Impregnation Method for Basal Membranes in Renal Biopsies

Silver impregnation methods are essential in biopsy interpretation in nephropathology with regard to visualizing the basal lamina and its associated changes. The most widely used methods, mainly Jones methenamine impregnation, are time-consuming in their protocols and require multiple microscopy control points. In this report, we propose an alternative, modified method for silver impregnation with methenamine solution with a significantly shorter protocol time and good staining quality, allowing for proper interpretation of basal lamina changes in the glomeruli and blood vessels. Furthermore, unlike some other modified techniques, our proposed protocol does not include microwaving of the solutions but rather a thermostat is used, thereby reducing fire hazards. Implementing the protocol in our everyday practice has reduced sample processing time while not negatively impacting biopsy interpretation.


Introduction
Renal biopsies are a standard pathological method with significant methodological specifics [1]. Interpretation of the basal membranes and their characteristics is critical for distinguishing between several types of glomerulonephritis, such as stage membranoproliferative and membranous nephropathy [1]. A suitable method for the visualization of the basement membranes of the glomeruli in renal biopsies and one of the most widely available methods for silver impregnation is the Jones silver stain, also referred to as methenamine periodic acid-Schiff (PAS) stain [2,3].
In this report, we propose a modified method of silver impregnation, suitable both for glomerular capillary basement membranes and tubular basement membranes.

General safety precautions
Technicians should wear safety gloves, goggles, and laboratory clothing covering all exposed skin areas. Inhalation and direct contact with the reagents should be avoided, especially with sodium thiosulfate, which is toxic if swallowed, and highly irritating to the skin, eyes, and respiratory tract; silver nitrate is highly irritating to the skin, eyes, and the gastrointestinal system, and is also a probable carcinogen.
Do not use metal instruments to avoid silver mirror reaction, depleting the silver in the working solution and decreasing staining reaction quality due to low silver deposition.

Tissue fixation
Fresh tissues should be fixated in a solution prepared on the spot, consisting of 1 gram of picric acid, 150 ml of 80% ethyl alcohol, and 60 ml of 40% formalin. For proper fixation of core needle biopsy specimens, tissues should remain in the fixative for 12-20 hours.

Slide preparation
Embed the fixated tissues in paraffin, cut them into 2 µm-thick sections using a conventional microtome, and fix them on glass slides.
Required reagents for staining are as follows:

Expected results and control tissues
Sections from healthy kidneys are optimal for staining control. Basement membranes stain in brownishblack ( Figure 1A) and elastic membranes of arterial vessels stain in black ( Figure 1B). The method is also specific and sensitive enough to distinguish basement membrane changes in glomerulonephritis showing good differentiation in tram-tracking for membranoproliferative glomerulonephritis (Figure 2A) and perpendicular basement membrane projections (spiking) and vacuoles in membranous nephropathy ( Figures  2B, 2C). The staining method is nonspecific for amyloidosis ( Figure 3) and, while again nonspecific, underlines the changes well in monoclonal immunoglobulin deposition ( Figure 4A), diabetic nephropathy class two ( Figure 4B) and focal segmental glomerulosclerosis ( Figure 4C), where proteinaceous deposits stain in black. Again, a nonspecific but good distinguishing potential is seen in thrombotic microangiopathy, where fibrin stains in brick brown ( Figure 4D).

FIGURE 1: Control staining and expected results
A: glomerular capillary basement membranes stained in brownish-black (arrow), modified silver impregnation, original magnification x400; B: elastic membrane of artery stained in black (arrow), modified silver impregnation, original magnification x400

FIGURE 2: Effectiveness of the staining method in distinguishing glomerular basement membrane changes
Tram-track sigh (arrow) in membranoproliferative nephropathy (A), spiking (arrow) and basement membrane vacuoles (arrows) in membranous nephropathy (B and C); modified silver impregnation, original magnifications x1000

Discussion
The proposed methods use a suitable fixator for the tissues, allowing for optimal histochemical staining with other stains, immunohistochemistry, genetic analysis, and electron microscopy. Other fixatives, such as neutral 10% formalin, lead to a marked contraction of dense fabric and low contrast in staining [1]. Our proposed modified silver impregnation is a sensitive method for visualization of tubular basal membranes, glomerular capillary basement membranes, and elastic fibers in the walls of small arteries, as well as subintimal fibrosis. The resulting stained tissue sections are of high quality and have good contrast.
Most readily available silver impregnation methods include a counterstain, which requires more reagents, prolongs the staining time, and allows for other mistakes in these protocols [4,5]. Furthermore, the slides stained in such a manner do not give any additional information about tissue pathology. Other than minimizing the reagents and hence the cost and possibility of errors in the staining protocol, our proposed method is significantly shorter in the time needed to carry out the methodology when compared to the most widely used silver impregnation method in nephropathology -the Jones silver stain [2]. Furthermore, some modifications of the Jones method include microwaving the slides in the solution in a loosely covered Coplin jar, which is a hazard for vapor production and explosion if the jar lid slips and seals under boiling temperatures [1]. The Jones method uses multiple microscopy control points in the protocol, reaching upwards of 10 for a single slide, and may require de-staining with potassium ferricyanide, which is a mild irritant but may produce highly toxic hydrogen cyanide vapors in acidic environments as used in the protocol [2]. In this aspect, our protocol not only involves significantly fewer steps and, therefore, faster, but also has fewer control points, no more than two per slide, which are several seconds from one another and not in between 10 minutes, thereby further reducing technician exposure to potential irritants and toxins. As seen in the provided figures, the reaction is easy to control and provides good staining results, adequate for differentiating basal membrane changes. Furthermore, unlike some modifications of the Jones procedure, our proposed protocol does not include microwaving of the solution and slides, which, depending on microwave type and power, can produce varying results while also being a fire and explosion hazard [4].

Conclusions
The proposed staining methodology, while not differing significantly from the established silver impregnation methods in nephropathology, has several advantages. The first of these is the significantly shortened staining time, minimal control points for microscopy, and equivalent staining results regarding basal lamina differentiation. In addition, thermostat incubation reduces the risks of fire hazards. The only way in which the visualization differs significantly is the lack of counterstaining, which does not reduce the