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6 protocols using gb27303

1

Multimarker Immunohistochemical Analysis

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Frozen slides were brought to room temperature, fixed in cold acetone for 10 min, then air dried. Samples were incubated in proteinase K working solution at 37°C for 25 min. Next, permeabilize working solution was used to cover objective tissue, then incubate at room temperature for 20 min. The slices were equilibrated at room temperature: The following kits and antibodies were used for staining: TUNEL Assay Kit (G1501, Servicebio, China), rabbit anti-KI67 antibody (1:200; GB121141, Servicebio, China), goat anti-CD3 antibody (1:200; GB111337, Servicebio, China), 488 anti-goat (1:400; GB25303, Servicebio, China) and Cy5 anti-rabbit (1:400; GB27303, Servicebio, China). The slices were washed three times with PBS (pH 7.4). Throwed away liquid slightly, then coverslip with anti-fade mounting medium. Images were acquired using an OLYMPUS laser scanning microscope.
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2

Quantifying Macrophage Polarization in Liver

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To evaluate macrophage polarization, formalin-fixed, paraffin-embedded liver sections were subjected to antigen retrieval in a microwave oven for 8 min in EDTA (pH 8.0), followed by blocking with peroxidase and serum. The sections were incubated overnight at 4°C with rabbit anti-CD163 antibody (GB13340, Servicebio, 1:3,000 dilution), followed by antibody detection with an HPR conjugated anti-rabbit antibody. The sections were then incubated with iF555-Tyramide (G1233, Servicebio, 1:500 dilution) at room temperature for 10 min, followed by heating in a microwave repair for 8 min in EDTA (pH 8.0). The sections were incubated with rabbit anti-CD86 antibody (19589S, CST, 1:2,000 dilution), followed by antibody detection with an HPR conjugated anti-rabbit antibody and incubation with iF488-Tyramide (G1231, Servicebio, 1:500). The sections were subsequently incubated overnight at 4°C with rabbit anti-F4/80 antibody (GB11027, Servicebio, 1:200 dilution), followed by antibody detection with a CY5 conjugated anti-rabbit antibody (GB27303, Servicebio, 1:400 dilution). The cell nuclei were counterstained with DAPI. M1 macrophages (F4/80+CD86+) and M2 macrophages (F4/80+CD163+) were counted in five randomly selected high power fields (200×) per sample, with the mean representing the infiltration of M1 and M2 macrophages.
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3

Immunofluorescent Analysis of Rat Femur Fracture

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Rat femur fracture specimens from six-week-old SD rats were obtained at 14 d, fixed for 24 h in 4% paraformaldehyde, embedded in paraffin, and then sectioned for histological analysis. The manufacturer's instructions were followed for staining various immunofluorescent proteins. The S100β (1:1000, GB12359; Servicebio, Wuhan, China) and β III-tubulin (1:1000, GB12139; Servicebio, Wuhan, China) specific primary antibodies were used in this work. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:400, GB25301, Servicebio, Wuhan, China) or cy5-conjugated goat anti-rabbit IgG (GB27303, Servicebio, Wuhan, China) was used as the secondary antibody and incubated at 25 °C for 2 h before being combined with DAPI for co-incubation at 25 °C.
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4

Multimarker Immunofluorescence Tissue Analysis

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This experiment followed conventional methodology [30 (link)], which included fixing fresh tissue, dehydrating, paraffin embedding, sectioning, mounting on slides, and incubating with primary antibodies (anti-α-SMA, green, 1:250, 14395-1-AP; anti-TGF-β1, red, 1:250, 21898-1-AP; anti-COL1A1, pink, 1:200, 67288-1-Ig. These antibodies were bought from Proteintech) and corresponding secondary antibodies in sequence (Alexa Fluor 488-conjugated secondary antibodies, 1:200, GB25303; CY3-conjugated secondary antibodies, 1:200, GB21303; CY5-conjugated secondary antibodies, 1:200, GB27303; these antibodies were bought from Servicebio), and DAPI was used for the counterstaining of nuclei (Servicebio, Wuhan, China). In this process, the sections were incubated with primary antibodies at 4 °C overnight. The images were acquired by Leica DM IL microscope (Confocal) (Leica microsystems, Wetzlar, Germany).
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5

Adipose Tissue Immunofluorescence Staining

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Adipose tissue sections were subjected to antigen retrieval by boiling in 10 mmol/L sodium citrate buffer (pH 4.5), followed by blocking with PBS containing 10% FBS and 3% BSA for 1 h. Next, the sections were incubated with an anti-Perilipin A antibody (A16295, Ablonal, Wuhan, China) overnight at 4°C. The slides were incubated with an anti-rabbit IgG conjugated with Cy3 (GB21303, Servicebio, Wuhan, China) or Cy5 red fluorescent dye (GB27303, Servicebio) at room temperature for 1 h, followed by TUNEL staining kits (G1504/G1502, Servicebio) according to the manufacturer’s instructions.
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6

Immunofluorescence Analysis of Mouse Testis

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The 5 μm paraffin sections from mouse testis were used for immunofluorescence assay. The experimental procedures were based on our previous study [14 (link)]. The antibodies against DDX4 (1:100, ab13840, Abcam, Cambridge, MA, USA), PNA-FITC (10 μg/mL) (Thermo Fisher, Waltham, MA, USA), CD68 (1:200, 28058-1-AP, Proteintech, Wuhan, China), and Cy5 conjugated Goat Anti-rabbit IgG (H+L) (1:500, GB27303, Servicebio, Wuhan, China) were applied here. The images were acquired under a fluorescence microscope (Zeiss, Jena, Germany).
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