Immunofluorescence analyses were performed as described previously15 (link),47 (link). Paraffin sections of fibrovascular tissues were deparaffinized and hydrated through exposure with xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10-mM citrate buffer (pH 6). Mouse eyeballs were fixed in 4% paraformaldehyde for 30 minutes on ice, incubated in PBS solution with increasing concentration of sucrose (10, 20, and 30%), and embedded in Frozen Section Compound (Leica, Exton, PA). Sections were probed with primary antibodies for galectin-1, AGE, CD45, CD68, GFAP, GS, Iba-1, IL-1β, IL1R1 and TLR4 followed by secondary antibodies. Nuclei were counterstained with DAPI, and sections were visualized under a Biorevo microscope (Keyence, Tokyo, Japan).
Biorevo microscope
Manufactured by Keyence
Sourced in Japan
The BIOREVO microscope is a high-performance microscope designed for biological research and analysis. It features a compact and ergonomic design, advanced optical systems, and user-friendly controls. The BIOREVO microscope is capable of providing clear, detailed images of biological specimens, enabling researchers to conduct detailed observations and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Frozen section compound, by Leica (1 mentions)
Rabbit anti glial fibrillary acidic protein, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Lonza (1 mentions)
Mouse anti gfap, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Mouse anti αb crystallin, by Abcam (1 mentions)
Rabbit anti phospho ser59 αb crystallin, by Abcam (1 mentions)
Rabbit anti at1r, by Santa Cruz Biotechnology (1 mentions)
Envision hrp kit, by Agilent Technologies (1 mentions)
Rabbit anti p rr, by Merck Group (1 mentions)
Image pro software, by Media Cybernetics (1 mentions)
Anti cd31, by Agilent Technologies (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti vegfr2, by R&D Systems (1 mentions)
Anti gfap, by Leica (1 mentions)
Anti galectin 1, by Abcam (1 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Bz 9000, by Keyence (1 mentions)
Ab diluent, by Agilent Technologies (1 mentions)
Ab155938, by Abcam (1 mentions)
12 protocols using biorevo microscope
1
Immunofluorescence Analysis of Ocular Tissues
2
Immunostaining of Ocular Tissues in SDT Rats
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For SDT fatty rats’ eyes, eyeballs were fixed in 4% paraformaldehyde. Paraffin sections of tissues were deparaffinized and hydrated through exposure to xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6.0). For antibody staining, sections were washed with PBS containing 0.05% Triton X-100 and 5% FBS. Sections were probed with the following primary antibodies: mouse anti-αB-crystallin (1:200; Abcam), rabbit anti-phospho Ser59-αB-crystallin (1:100; Abcam), rabbit anti-glial fibrillary acidic protein (GFAP; 1:100; DAKO, Carpinteria, CA, USA), mouse anti-GFAP (1:100; Thermo Fisher Scientific), and rabbit anti-IL-1R (1:100; R&D Systems) antibodies. The secondary antibodies for fluorescent detection were Alexa Fluor 488 (1:200; Abcam) and 546 (1:200; Thermo Fisher Scientific). Nuclei were counterstained with DAPI (1:100; Lonza), and sections were visualized using a Biorevo microscope (Keyence).
3
Immunohistochemical Analysis of (P)RR and AT1R
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The slides were pre-incubated with normal goat serum to block non-specific binding, followed by peroxidase block solution to block endogenous peroxidase activity. Sections were probed with rabbit anti-(P)RR (Sigma-Aldrich) and rabbit anti-AT1R (Santa Cruz Biotechnology) primary antibodies. Color was developed using the Envision HRP kit (DAKO, Carpinteria, CA, USA). Normal rabbit IgG was used as a negative control antibody. Sections were analyzed using the BIOREVO microscope (Keyence).
4
Quantification of Cardiac Fibrosis
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Heart tissues were fixed with 4% formalin and embedded in paraffin. Five-μm-thick sections were prepared and stained with hematoxylin and eosin or Masson’s trichrome stain. For measurement of cardiac fibrosis area, the high-resolution images (×100 magnification) of the heart sections stained with Masson’s trichrome were taken using a BIOREVO microscope (BZ9000; Keyence) and fibrosis area was quantified using the Image-Pro software (Media Cybernetics).
5
Immunohistochemical Analysis of Fibrovascular Tissues
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Paraffin sections of fibrovascular tissues were deparaffinized and hydrated through exposure with xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6). Sections were probed with the following primary antibodies: anti-Galectin-1 (Abcam), anti-VEGFR2 (R&D systems, Minneapolis, MN), anti-CD31 (DAKO, Tokyo, Japan), and anti-GFAP (glial fibrillary acidic protein; Leica, Exton, PA) antibodies. The secondary antibodies for fluorescent detection were AlexaFluor 488 and 546 (Life Technologies). Nuclei were counterstained with DAPI (diamidino-2-phenylindole), and sections were visualized under a Biorevo microscope (Keyence, Tokyo, Japan).
6
Immunohistochemical Analysis of Rac and p-Src
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Immunohistochemistry was used to measure the levels of Rac and p-Src in the cells. After PBS washing, rehydrated culture dishes were incubated with primary antibodies against Rac (ab155938) and p-Src (ab40660) purchased from Abcam (Tokyo, Japan) diluted at 1:100 in Ab Diluent (Dako ChemMate; Dako, Japan) overnight at room temperature. For staining with Alexa Fluor 488 anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA), secondary antibodies were diluted at 1:200 in Ab Diluent and added for 60 min at room temperature in the dark. Digital images were taken on a BIOREVO microscope equipped with a confocal microscopy system (BZ-9000, Keyence, Japan) as described previously28 (link).
7
Immunofluorescence Staining of Adherent Cells
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The cells were grown on a poly-l -lysine–coated coverslip. For immunostaining, the cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4) for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. The fixed cells were blocked with 3% bovine serum albumin (BSA)/PBS for 1 hour at room temperature. Then, the cells were incubated with the primary antibodies diluted with 3% BSA/PBS for 1 hour in a humidity box, stained with Alexa Fluor–conjugated secondary antibodies for 1 hour, counterstained with DAPI for 5 min, and mounted with VECTASHIELD (Vector Laboratories). Images were acquired using a BioRevo microscope (BZ-9000; Keyence) equipped with a 40× objective lens [Plan Apochromat, numerical aperture (NA) 0.95, Nikon] and a 100× oil objective lens (Plan Apochromat VC, NA 1.4). Cellular counting and image cropping were performed using ImageJ.
8
Spinal Cord Cytoarchitecture Analysis
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Z-stack Images of the DRG and spinal cord were digitally photographed using a Keyence BIOREVO microscope (BZ-9000, Keyence, Osaka, Japan) and transferred to Photoshop CS6 to generate the figures. Some sections were also examined by confocal laser scanning microscopy (FV 1000D; Olympus, Tokyo, Japan) to construct the 3D images. The number of BDA-positive and BDA/CGRP double-positive DRG neurons of intact P40 (n = 4) were counted, and the percentage and standard deviation of BDA/CGRP double-positive neurons per BDA-positive DRG neuron were calculated. The percentage and standard deviation of BDA/RT-97 double-positive neurons per BDA-positive DRG neuron were calculated in the same manner. Histological analysis was performed in four animals of each group.
The cytoarchitecture of the L6 segment was determined by referring to a spinal cord atlas (Sengul et al., 2013 ). We identified the IML by referring to the results of the retrograde tracing study, as well as to the atlas showing ChAT immunoreactivity in the spinal cord (Nadelhaft and Booth, 1984 (link), Sengul et al., 2013 ). Although BDA-positive primary afferent fibers projected to the ventral horn, we analyzed the projections to the dorsal horn (DH), and intermediate zone (IZ).
The cytoarchitecture of the L6 segment was determined by referring to a spinal cord atlas (Sengul et al., 2013 ). We identified the IML by referring to the results of the retrograde tracing study, as well as to the atlas showing ChAT immunoreactivity in the spinal cord (Nadelhaft and Booth, 1984 (link), Sengul et al., 2013 ). Although BDA-positive primary afferent fibers projected to the ventral horn, we analyzed the projections to the dorsal horn (DH), and intermediate zone (IZ).
9
Sphere Formation Assay Protocol
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For analysis of sphere formation, second-generation spheres were thoroughly trypsinized and sieved through a cell strainer with 40-μm nylon mesh (Thermo Fisher) to prepare a single-cell suspension. Cells were suspended in serum-free media (SFM) composed of DMEM/F12 (Gibco), 20 ng/ml EGF, 10 ng/ml bFGF, 10 ng/ml IGF, 2% B27 supplement (Gibco), and 10% Wnt3a-CM, and 200 cells per well were seeded in ultra-low attachment 24-well culture plate (Corning). Varying concentrations of antibodies were added and the cells were fed twice a week by 100 μl SFM. After 14-day incubation, 63 fields in each well were scanned and merged using BIOREVO microscope (BZ-9000; Keyence) to display the whole well image. The number of tumor spheres (>100 μM) was counted or measured from the images. To determine total number of viable sphere-forming cells, spheres were collected by centrifugation and dissociated into single-cell suspension by trypsinization. Cells were washed twice with PBS, stained with 2 μM Calcein-AM for 1 h, and analyzed by flow cytometry.
10
Immunofluorescence Analysis of Murine Kidney
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Mouse kidneys were collected at 28 days after ADR administration. Renal tissues were frozen with liquid nitrogen in OCT compound immediately after collection. Frozen tissues were sliced into 10-μm thickness using cryostat and attached onto glass slides. Sections were thawed with ice-cold acetone at − 20 °C for 5 min. After blocking with Protein Block Serum-Free (Dako, USA) at room temperature for 30 min, sections were reacted with primary antibody diluted at 1:100 and secondary antibody diluted at 1:300 with Antibody Diluent with Background Reducing Components (Dako, USA). Primary antibody was reacted overnight at 4 °C and secondary antibody was reacted for 1 h at room temperature. Polyclonal rabbit anti-WT-1 (sc-192; Santa Cruz Biotechnology), purified rat anti-mouse CD44 (#550538; BD, USA), Goat anti-rabbit IgG (H + L) Alexa Fluor 488 (A-11008; Thermo Scientific Inc., USA) and Goat anti-rat IgG (H + L) Alexa Fluor 488 (A-11006; Thermo scientific Inc., USA) were used for antibody reactions. Sections were incubated with 1 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) solution for 20 min at room temperature. Then, stained sections were mounted with VECTASHIELD mounting medium (Vector Laboratories, USA). Images were captured using BIOREVO microscope (Keyence, Japan).
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