N histofine mousestain kit

Tissue sections were deparaffinized and subsequent blockage of the endogenous peroxidase activity was achieved by incubation with 2.5% methanolic hydrogen peroxide for 30 min. The tissue sections were stained with rabbit antibodies against Ki-67, MMP13, collagen I, desmin (GeneTex, Irvine,CA) or TIMP-1 (Proteintech Group Inc, Chicago, USA) by using the Mouse/Rabbit Probe HRP Labeling Kit (BioTnA Biotech, Kaohsiung, Taiwan). Binding of mouse monoclonal antibodies against GNMT (14-1, YMAC Bio Tech, Taiwan), α-SMA (Lab Vision, Fremont, CA) and TGF-β1 (R&D Systems) was detected by using the N-Histofine Mousestain Kit (Nichirei Biosciences, Tokyo, Japan). The immunohistochemical assays were performed according to the manufacturer’s instructions. The sections were developed with 3-3′ diaminobenzidine (DAB) (Dako, Milan, Italy) and finally counterstained with hematoxylin (Dako). Negative controls were performed using normal mouse antiserum instead of the primary antibody.

After sacrificing, tumor tissues were immediately harvested and fixed in 10% phosphate buffered formalin. The tissues were embedded in paraffin and sectioned (8 μm), followed by hematoxylin and eosin (H&E) staining. Paraffin-embedded tumor tissues were stained for proliferating cell nuclear antigen (PCNA) using N-Histofine^® MOUSESTAIN KIT (Nichirei Biosciences Inc., Tokyo, Japan) following the manufacturer’s protocol [62 (link)]. The primary antibody was mouse monoclonal anti-PCNA antibody (Abcam ab29).
To evaluate the systemic toxicity of GOFA-DOX/HACPN, major organs including hearts, livers, spleens, lungs and kidneys were harvested before euthanasia and embedded in paraffin and sectioned for H&E staining. Blood samples were collected for hematologic analysis (white blood cell count, red blood cell count, hemoglobin and hematocrit) and biochemical analysis (aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen and creatinine) of major organ functions. The mice in the control group (saline) were used as comparison.

For immunohistochemical analysis, kidney tissues from the sacrificed mice were harvested and fixed in 10% formaldehyde. Paraffin cross-sections with a thickness of 5 μm were stained with antibodies against MT-I/II (Invitrogen), followed by N-Histofine^® MOUSESTAIN KIT (414321F; Nichirei Biosciences Inc.; Tokyo, Japan). To measure renal fibrosis, the paraffin-embedded sections were stained using Masson’s Trichrome stain kit (ScyTek Laboratories Inc., Logan, USA). Quantification of kidney immunohistochemical assay and fibrosis on Masson’s trichrome-stained sections were performed using ImageJ analysis. The raw pixel values of the selected areas were estimated using the ImageJ software. These values indicated the distribution area times the stain intensity of the images.

GFP plasmids were mixed with jetPEI and intratracheally delivered into 8-week-old female C57BL/6 mice. The lungs were harvested from each mouse 48 hours later; then, they were fixed with 3% paraformaldehyde solution for 16 to 18 hours and embedded in wax. Samples were cut into 4-μm slices for immunohistochemical staining with mouse anti-GFP antibody (Millipore MAB3580) (Millipore, Billerica, MA, USA) and the N-Histofine® MOUSESTAIN KIT (Nichirei, Tokyo, Japan). The distribution of the GFP protein was observed under light microscopy and photographed (Zeiss, Germany) (Fig 5).