Tissue sections (4-μm) from formalin-fixed, paraffin-embedded tumor xenografts were immunostained with anti-Ki67 and –active caspase-3 antibodies (1:100 dilution) using the MaxVision kit (Maixin, Fuzhou, China) according to the standard protocol as described [23 (link), 28 (link)]. Color was developed with 0.05% diaminobenzidine and the sections were counterstained with hematoxylin. The sections were mounted with Permount™ Mounting Medium and photographed under an inverted fluorescence microscope.
Maxvision kit
Manufactured by Maixin Group
Sourced in China
The MaxVision kit is a versatile laboratory equipment designed for performing a variety of scientific tasks. It includes essential components for various experiments and analyses. The core function of the MaxVision kit is to provide the necessary tools and accessories to support diverse research and testing activities in a laboratory setting.
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7 protocols using maxvision kit
1
Immunohistochemical Analysis of Tumor Xenografts
2
Quantifying Spleen Cell Proliferation and Apoptosis
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Mice were humanely euthanized, their spleens extracted, and the tissues were then fixed using formalin, embedded in paraffin, and sectioned (5 μm thickness) for subsequent staining. As per the manufacturer’s directive, sections were stained with anti-Ki67, anti-Active-Caspase-3, and anti-PSAT1 antibodies utilizing the MaxVision kit (Maixin, Fuzhou, China). Diaminobenzidine (0.05%) was the chosen chromogen, and hematoxylin served as the counterstain. Prepared sections were then mounted using Permount™ Mounting Medium and visualized under inverted fluorescence microscopy.
3
Histological and Immunohistochemical Analysis of Rabbit Knee Joints
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After the rabbits were sacrificed, the knee joint samples were removed and fixed in 10% neutral buffered formalin for 3 days and were then dehydrated through an alcohol gradient (30%–100%) and embedded in paraffin wax. The specimens were cut to a thickness of 5 μm along the longitudinal axis of the tendon. These sections were treated with haematoxylin and eosin (H&E) and Masson’s trichrome stain (Masson’s) for histological evaluation. The sections stained with H&E and Masson’s were observed using a light microscope (Eclipse 80i, Nikon), and the images were captured using a camera (DS-5M, Nikon). For immunohistochemistry staining, endogenous peroxidase was blocked by incubation with 3% (v/v) hydrogen peroxide in methanol for 10 mins. Then, the sections were blocked for 20 mins with a blocking reagent containing goat serum in PBS after washing three times with PBS. After overnight incubation at 4°C with a primary antibody, the sections were washed and then incubated with a secondary antibody (MaxVision kit; Maixin Biotechnology) for 15 mins at room temperature. Dimethyl aminoazobenzene (DAB) (Simple Stain DAB Solution, Maixin Biotechnology) was used for colour development. Hematoxylin staining was used to reveal the nuclei. The results were evaluated by three individuals who were blinded to the treatments.
4
Immunostaining of Tumor Xenografts
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Formalin-fixed tissues were embedded in paraffin and sectioned. Tumor xenograft sections (4.0 μm) were immunostained with Ki67 and c-ABL antibodies using the MaxVision kit (Maixin, Fuzhou, China) according to the manufacturer’s instructions37 (link).
5
Quantification of hTERT Expression
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SW480 cells were plated on 2.5 cm coverslips at a density of 5×105 cells/well in 6-well plates. After transfection for 24, 48, or 72 h, coverslips were removed and immunostained with the rabbit polyclonal anti-hTERT antibody (1∶300, Santa Cruz Biotechnology, CA, USA) and the Maxvision kit (Maixin, Fuzhou, China). On each coverslip, five views were randomly selected and captured using image analysis software (Leica, Germany). In each view, the grayscale intensity was measured at 10 randomly selected points and averaged. The average grayscale intensity was inversely proportional to the protein level.
6
Immunohistochemical Analysis of Collagen I
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The sections of PET artificial ligament in the articular cavity were deparaffinized and rehydrated. Endogenous peroxidase was blocked for 30 min with 0.3% hydrogen peroxide at 37 °C, and further for 20 min with a blocking reagent (PBS containing goat serum). The sections were washed twice with PBS buffer and incubated with the primary antibody (rabbit anti-dog COL1, dilution 1: 50, Bioworld technology co Ltd., Nangjing, China) at 4 °C overnight. After washing, the sections were incubated with a secondary antibody (MaxVision kit, Maixin Biotechnology, Fuzhou, China) for 15 min at room temperature, and then treated with dimethylaminoazobenzene (Simple Stain DAB Solution, Maixin Biotechnology, Fuzhou, China) for 5 min, followed by counter-staining with hematoxylin. A DP Manager (Olympus Optical Co., Tokyo, Japan) was used to obtain the digital images, and Image-Pro Plus 6.0 software (Media Cybernetics Corp., Rockville, USA) was used to measure COL1 expression, which was expressed as the mean area value of COL1-positive staining at the graft site with 200× magnification.
7
ERCC1 Protein Expression Evaluation in Formalin-Fixed Paraffin-Embedded Tumor Specimens
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Formalin-fixed, paraffin-embedded tumor specimens obtained by biopsy were cut into 4-µm sections. Slides were deparaffinized in xylene for epitope retrieval, exposed to 10 mM citric acid antigen retrieval solution (pH 6.0), and heated for 30 min in a water bath. PBLs were fixed with methanol and Triton X-100 was used to permeabilize membranes.
Specimens were incubated overnight with a monoclonal antibody against human ERCC1 protein (mouseclone 8F1, Maixin Bio, Fujian, China). Antibody binding was detected with the Maxvision kit (sheep/rabbit anti-mouse, Maixin Bio, Fujian, China) by incubation for 15 min with the substrate and counterstained with hematoxylin.
Two pathologists who were unaware of clinical data independently evaluated the percentage of positive tumor nuclei under a light microscope at a magnification of 400X. The grading system was as follows: immunoreactivityin ≥10% of tumor cells or PBLs was considered as positive. If <10%, it was defined as negative (Wachters et al., 2005) .
Specimens were incubated overnight with a monoclonal antibody against human ERCC1 protein (mouseclone 8F1, Maixin Bio, Fujian, China). Antibody binding was detected with the Maxvision kit (sheep/rabbit anti-mouse, Maixin Bio, Fujian, China) by incubation for 15 min with the substrate and counterstained with hematoxylin.
Two pathologists who were unaware of clinical data independently evaluated the percentage of positive tumor nuclei under a light microscope at a magnification of 400X. The grading system was as follows: immunoreactivityin ≥10% of tumor cells or PBLs was considered as positive. If <10%, it was defined as negative (Wachters et al., 2005) .
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