Picrosirius Red Staining for Semiquantitative Histopathologic Evaluation of Collagen Deposition in Murine Models of Chronic Lung Allograft Rejection

Our research focuses on analyzing the development of fibrosis in chronic lung allograft rejection. We are therefore interested in methods that characterize the fibrotic tissue compartment. To reproduce transplant-related diseases, we employ the mouse model of single lung transplantation that we introduced several years ago.

In this model, the transplantation of a lung between allogeneic mouse strains has been shown to result in different levels of graft fibrosis. Histology data on fibrotic tissue remodeling is often presented qualitatively. Our method using Picrosirius Red staining offers the advantage of a semi-quantitative evaluation and differentiates between thick and thin collagen fibers.

To begin, hydrate the paraffin-embedded mouse lung samples in decreasing ethanol concentrations for two minutes each. Stain the slide for eight minutes in Meyer's hemalum solution. Wash the slide for 10 minutes under running tap water, then incubate the slide for one hour in Picrosirius Red solution.

Dip the slide five to 10 times in 0.5%acetic acid solution. Gently shake the slide to remove the solution. Now dehydrate the slide in increasing ethanol concentrations and 100%Xylol for five minutes each.

Add one drop of mounting medium onto the sample for embedding and cover with a cover slip. Allow the slides to dry under a fume hood. Under a light microscope, use appropriate imaging software to focus the sample until a clear and sharp image is obtained.

Adjust the degree of polarization filter until the background is completely dark or black. Scan the sample under 20x magnification and export the images in TIFF format. Transfer the images to Fiji software.

Click Edit and select Clear Outside. Then click Analyze followed by Measure to measure the total surface area. To measure total collagen content per sample, click Image followed by Adjust and Color Threshold.

Set the hue between 2 and 130 and brightness between 35 to 255. Then click Analyze and, in the Analyze Particles window, set the size to 1 minus infinity and tick the Clear and Summarize boxes. Adjust the hue to red for the analysis of thick collagen fibers and yellow green for thin collagen fibers, and measure the total collagen count.

Finally, click Plugins, Macros, and Record to configure the macro for automated processing. Masson's trichrome, Herovici's and Picrosirius Red staining illustrated extensive parabronchial and paravascular collagen deposition in the major histocompatibility complex mismatched model. The minor mismatched model primarily showed dense lymphocytic infiltrates with less intense collagen deposition.

Picrosirius Red staining with a polarization filter revealed a green appearance of the deposited fibers across both models. Isograft controls showed collagen restricted to parabronchial and paravascular borders, similar to naive murine lungs. Digital image analysis revealed increased total collagen in major histocompatibility complex mismatched lung grafts compared to minor mismatched grafts, right-sided naive lung tissue, or naive BALB/c left lungs.

No differences in thick collagen fibers were demonstrated. Analysis of thin collagen fibers showed increased presence in the major mismatched model compared to the minor mismatched model, naive right-sided lung tissue, or naive BALB/c left lungs.