Modified PASM-Masson Staining in a Renal Biopsy

Summary

In this study, we present a protocol of PASM-Masson dual staining to identify the location and extent of glomerular immune complex deposition in renal diseases.

Abstract

Special histochemical staining is an important method for a renal biopsy, which includes PAS staining, periodic acid silver methenamine (PASM) staining, and Masson staining. In this study, we aim to develop and modify the method of PASM and Masson double staining (PASM-Masson) in a renal biopsy. One hundred and fifty-two cases of renal biopsy were detected by PASM-Masson staining in our hospital. The results showed that the background of the slices was clean, and the renal structures were more visible under the microscope. The glomerular basement membrane, renal tubular basement membrane, and capillary basement membrane were stained black or brown; the mesangium and collagen fibers were stained blue black; the renal tubular epithelial cells and immune complexes were dyed red. These findings indicate that the glomerular immune complex deposition can be accurately identified and positioned by modified PASM-Masson staining, which might have important effects on the pathological diagnosis of renal diseases.

Introduction

Special histochemical staining has been used by pathologists to assist in tissue-based diagnoses for over 100 years^1,2. The application of special staining in the diagnosis of renal diseases has been recognized as an important method for distinguishing the types of renal diseases and displaying some special components in renal tissues³.

To date, a variety of special stains have been applied to renal biopsies, which includes periodic acid silver methenamine (PASM) staining and Masson's trichrome staining. Lesions of the glomerular capillary walls are the main pathological changes, which helps to characterize and differentiate various glomerular diseases. PASM staining is mainly used to observe the degree of abnormalities of the glomerular capillary walls in glomerular basement membrane^4,5. Masson staining is utilized to investigate an extremely wide variety of glomerular diseases^6,7 and has been found to be effective in identifying the accumulation of the immune complex as well as the proliferation of basement membranes and renal interstitial collagen fibers⁸. For example, chronic graft damage in both the medulla and the cortex in renal transplant biopsies was measured by PASM and Masson histochemistry, respectively⁹. However, if PASM and Masson staining are done separately, it is difficult to accurately detect the localization of the basement membrane and immune complex, and to judge whether these two lesions exist at the same site. On the contrary, PASM-Masson dual staining can clearly distinguish the staining and localization of the basement membrane and the immune complex in the same section. Moreover, it can shorten the staining time and improve the work efficiency. Therefore, PASM-Masson staining plays an important role in the diagnosis of glomerular diseases.

In order to identify the location and extent of glomerular immune complex deposition in different types of renal diseases, 152 cases of renal biopsy were collected and PASM-Masson dual staining was performed in the present study.

Protocol

The protocol follows the guidelines of our hospital's human research ethics committee. One hundred and fifty-two cases of renal biopsy specimens were collected from the Department of Pathology at the Shenzhen Third People's Hospital (The Second Affiliated Hospital of Southern University of Science and Technology) from January 2018 to May 2019.

1. Material preparation

1. Fix all tissues with 4% neutral buffered formaldehyde (pH 7.0) for 24 h.
2. Dehydrate the fixed tissues in an ethanol series (100%, 95%, 80% and 75%) for 1 min in each percentage, clear in xylene and then embed in paraffin wax.
3. Cut the slices into 1-1.5 µm slices and incubate for 40 min in a 70 °C oven.
   NOTE: The main chemical reagents include Bouin's fixed solution (saturated picric acid: formaldehyde: glacial acetic acid = 15: 5: 1), Masson kit (iron hematoxylin, bright red acid fuchsin working fluid, aniline blue, phosphomolybdic acid), and Periodic acid-sliver methenamine (PASM) kit (periodate, borax solution, silver nitrate hexamethylenetetramine powder).

2. Staining

1. After preparing the tissues in step 1, fix the tissues immediately in 10% formalin.
   NOTE: The 1-1.5 µm thick tissue sections are strongly recommended.
2. Deparaffinize each slice in dimethylbenzene for 5 min and then repeat once. Oxidize the slices with 1% periodate for 30 min and rinse with distilled water for 5 s.
3. Immerse the sections into hexamine silver (working solution) for 35 min (water bath 70 °C). After the color turns golden yellow, terminate the staining and wash the slides with distilled water for 5 s.
   NOTE: The reaction time can be adjusted according to the temperature of Bouin's solution, hexamine silver working solution, and Masson solution (Bouin's solution, 60 °C water bath for 1 h, 37 °C for 4 h and 26 °C for 24 h; Hexamine silver solution, 70 °C for 35 min and 60 °C for 50 min; Masson solution, 37 °C for 20 min and 26 °C for 30 min).
4. Decolorize the sections with 100 µL of 0.1% gold chloride for 1 min and observe the staining under light microscopy. When the golden-yellow staining disappears and basement membranes show an obvious black staining, wash the sections with distilled water three times.
   NOTE: The optimal time of PASM-Masson staining is the critical step in the protocol. The staining needs to be controlled under light microscopy.
5. Fix the tissues with 3% sodium thiosulfate for 1 min, replenish in Bouin's solution (37 °C water bath for 4 h) and wash the sections with distilled water for 5 min.
6. Stain the sections with Mayer hematoxylin solution for 15 min, and incubate in 45 °C water for 30 s.
7. Dye the specimens with 100 µL of Masson solution for 30 min and rinse with distilled water for 5 s. Differentiate the sections with 100 µL of 1% phosphomolybdic acid for 5 min.
8. Remove and shake off the phosphomolybdic acid by hand. Dye the sections with 100 µL of 1% aniline blue for 5 min.
9. Rinse the sections with distilled water. Fix with 1% acetic acid for 2 min.
10. Dehydrate the sections lastly by 95% for 10 s and then 100% alcohol for 1 min. Seal.

Results

PASM-Masson staining before modification (Figure 1A-D) showed that the hexamine silver staining was dyed deep black, and Masson staining showed a light dyeing on the immune complex in the glomerulus. In addition, the contrast between the basement membrane and the immune complex was not distinct. As a whole, the structures of the glomeruli and renal tubules were indistinct, and the background colors displayed hyperchromasia.

Discussion

Pathological diagnosis is an important tool for the diagnosis and differential diagnosis of renal diseases, and it is of great significance for the guidance of treatment and predicted value of renal diseases^10,11,12. Special staining is an essential method for renal biopsy including PAS staining, PASM staining and Masson staining^3,13,14...

Materials

Name	Company	Catalog Number	Comments
Absolute ethyl alcohol	Guanghua company (Guangzhou city, China)	1.17113.023
Bouin's fixed solution	Kaixiu company (Guangzhou city, China)	20180627	Sealing
Dimethylbenzene	Sinopharm Chemical Reagent limited corporation, China	20180607
Ethyl alcohol (75%)	Likang company (Nanchang city, China)	20180915
Ethyl alcohol (95%)	Likang company (Nanchang city,China)	20181005
Glacial acetic acid	Lingfeng company (Shanghai city, China)	20170912
Hematoxylin staining solution	BASO company (Zhuhai city, China)	718101
Masson kit	Weigxi company (Guangzhou city, China)	18121101	Store at 4°C
PASM kit	BASO company (Zhuhai city, China)	618091	Store at 4°C