Advantages of Sirius Red Staining for Quantitative Morphometric Collagen Measurements in Lungs

Abstract

Sirius Red staining is presented as a method for collagen determination, enabling quantitative morphometric measurements to be performed in locally defined tissue areas. The advantage of this method is especially shown for alveolar lung tissue. By excluding the bronchial areas in the tissue sections, the differences in the degree of fibrosis proved to be more discrete after different loads of quartz dust than by any other method. The difference of 12 micrograms collagen measured colorimetrically represented a 1.2-fold increase. The collagen measured in the alveolar tissue by the morphometric method rose from 9.8 to 28.6%. This is a 2.9-fold increase, underlining the vast improvement in sensitivity. Thus, this method is specifically suitable for the evaluation of very small fibrotic lesions. The quartz doses given are particularly low compared to most other investigations. Histologic lung and lymph node sections from female Wistar rats injected intratracheally with differing quantities of quartz dust (0.03, 0.1, 0.5, 1.75 mg) were stained with Sirius Red, and the collagen fibers measured with a quantitative image analysis. The results for lymph nodes using different methods (wet weight determination, quantitative measurement of quartz typical areas, colorimetric and morphometric collagen determination) showed a high correlation at the different doses. This showed that the morphometric method is suitable for the quantitative measurement of collagen. Corresponding results were also found in the comparative lung tissue measurements (colorimetric and morphometric collagen determination). However, the morphometric method has the decisive advantage that measurements can be restricted to defined tissue areas and do not destroy the section.

Brain-Specific Deletion of Extracellular Signal-Regulated Kinase 2 Mitogen-Activated Protein Kinase Leads to Aberrant Cortical Collagen Deposition

“…To provide more specific evidence of collagen deposition, brain sections were stained with Sirius Red, considered a specific collagen stain. 24,25 Parenchymal fibrils showed bright red fluorescence when viewed with polarizing filters ( Figure 5, A and B), a property suggested to be specific for type I collagen, compared with the green birefringence characteristic of type III (cartilagenous) collagens. 24 Electron microscopy was performed on portions of frontal cortex of 6-week-old ERK2 cKO mice as well as control littermates.…”

Anti-Vascular Endothelial Growth Factor Gene Therapy Attenuates Lung Injury and Fibrosis in Mice

“…Paraffin sections (3-m thick) were adhered to slides and stained with H&E. The pathological grade of inflammation and fibrosis in the whole area of the midsagittal section was evaluated under ϫ40 magnification and determined according to the following criteria: 0, no lung abnormality; 1, presence of inflammation and fibrosis involving Ͻ25% of the lung parenchyma; 2, lesions involving 25-50% of the lung; and 3, lesions involving Ͼ50% of the lung. Furthermore, the slides were stained with Sirius Red to assess the collagen deposition as previously described (12).…”

TIMP-1 Alters Susceptibility to Carcinogenesis

“…Images were captured at high magnification under normal illumination and polarized light on a Leica DM-RXA microscope attached to a Leica digital camera operated by OpenLab software (Improvision). Presence of mature fibrillar type I collagen was assessed from captured images (40,41) using the morphometric quantification technique described previously (42). For each field, an image taken under polarized light was quantified for pixel density using a set threshold for detection of the strongly birefringent signal indicative of fibrillar collagen.…”