Masson s trichrome stain

Rats were sacrificed on the 1st, 14th, and 28th day after surgery. Three rats in each group were sacrificed on the 1st and 14th day, and four rats were sacrificed on the 28th day. Then, the tendon of the injured site was collected. For histological analysis, tendons were fixed in 4% paraformaldehyde for 24 h. Sagittal paraffin sections were prepared by embedding the samples in paraffin and cutting into 4 µm thick sections. Four sections per sample were stained with HE and observed under the OLYMPUS EX-51 microscope (Tokyo, Japan) at 100 × magnification. Masson’s trichrome stain (Solarbio, G1340) was performed according to kit directions. RECA-1 is a cell surface antigen expressed by rat endothelial cells. In this study, RECA-1 was selected for immunohistochemistry analysis on days 1, 14 and 28. Then, 5-μm-thick continuous sections were incubated with an anti-RECA-1 antibody (abcam, ab22492, 1:1000) produced in rabbit followed by goat anti-rabbit immunoglobulin antibody conjugated by horseradish peroxidase. Then, slides were visualized using diaminobenzidine (DAB) substrate.

After 8 weeks, the BALB/c-nu mice were euthanized for histological examinations. The implants (n = 4) were taken out and fixed by 4% PFA for 3 days followed by decalcification for 12 days in 10% EDTA (pH 7.4). The specimens were dehydrated after decalcification and then embedded in paraffin. 5 mm sections (n = 6) were cut and stained with H&E or Masson's trichrome stain (Solarbio, China). The semiquantitative image analysis was performed by ImageJ (NIH Image, USA).

The animals were killed with an overdose of isoflurane (>5%). Their hearts were dehydrated and embedded after fixation with 4% paraformaldehyde. Paraffin sections were subjected to hematoxylin and eosin (H&E) staining and Masson's trichrome stain (G1340-7, Beijing Solarbio Science & Technology Co., Ltd.). Heart sections were subjected to immunohistochemical staining with an alpha-smooth muscle actin antibody (α-SMA, ARG66381), and then, development was performed with DAB as previously described [19 (link)].

Changes in renal morphology were examined in H&E and MASSON staining. The kidney tissues were fixed, fully automatic dehydrated, paraffin-embedded, and sliced into tissue sections (4 mm). All the slices were stained with hematoxylin for nuclei, and Masson’s trichrome stain (Solarbio, China) was performed according to the manufacturer’s specific instructions.

The right distal femur was harvested at each group (n = 6 knees/interval), fixed with 4% paraformaldehyde, decalcified with EDTA decalcifying solution (pH 7.2, E1171, Solarbio), and paraffin-embedded after conventional dehydration. The sections were created along the sagittal plane (6 μm), and we observed the femoral condyle cartilage and subchondral bone morphology. Safranin O fast green (Modified Safranin O Fast Green FCF Cartilage Stain Kit, G1371, Solarbio) was used to observe the morphological changes of the femoral condyle cartilage and subchondral bone. The Mankin score was used to evaluate articular cartilage changes [21 (link)]. Masson's Trichrome Stain (G1340, Solarbio) was used to evaluate cartilage collagen changes and calculate the collagen volume fraction (CVF). Representative sections of the tibiofemoral articular surface were photographed after observation under the microscope (Olympus BX43F Microscope), and the areas of interest were evaluated using IPP 6.0. The scores were calculated blindly by two researchers and averaged for statistical comparison.