Brains samples were fixed with 4% paraformaldehyde for 24 h and were transferred into 30% sucrose at 4°C for 12 h. Samples were then cryo-sectioned into 15 μm sections by microtome (CM1860; Leica) and dried at 60°C in an incubator (DHG-9101; SANFA, Yangzhou, China) for 4 h. Sections were then hydrated in 0.01 M PBS for 15 min, antigen retrieval was performed following manufacturer’s instructions (P0083, Beyotime, Beijing, China). Sections were further washed with 0.01 M PBS for three times, blocked with 0.1% FBS for 1 h at 37°C, and then incubated with a c-fos primary antibody (rabbit antimouse, ab190289, 1:1000; Abcam) for 2 h at 37°C and HRP (horse radish peroxidase) secondary antibody (PV-6001, 1:200; Zsbio, Tianjin, China) for 20 min at 37°C. Between incubation by antibody and after incubation by secondary antibody, sections were washed as described previously. A DAB kit (ZLI-9018; Zsbio) was used to develop the staining. Morphology was assessed using a light microscope (CX31; OLYMPUS). According to the mouse brain in stereotaxic coordinates (36 ), LPBN is located in the dorsolateral pons that surrounds the superior cerebellar peduncle (scp), which we considered as the neuroanatomical landmark to delimit LPBN in this work. By using ImagePro Plus 6 (Media Cybernetics, Inc. USA), the number of c-fos immunopositive neurons within LPBN were counted.
P0083
Manufactured by Beyotime
Sourced in China
The P0083 is a laboratory equipment used for electrochemical analysis. It measures the properties of electrochemical systems, including potential, current, and charge. The device is designed to be used in research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
A1933, by Merck Group (2 mentions)
Dfc700t, by Leica (2 mentions)
St795, by Beyotime (2 mentions)
Bl539a, by Biosharp (2 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Cm1860, by Leica (1 mentions)
Ab190289, by Abcam (1 mentions)
Zli 9018, by ZSGB-BIO (1 mentions)
Pv 6001, by ZSGB-BIO (1 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (1 mentions)
G8590, by Solarbio (1 mentions)
Image pro plus software program, by Media Cybernetics (1 mentions)
Dp71 microscope, by Olympus (1 mentions)
Ab181114, by Abcam (1 mentions)
P0260, by Beyotime (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti ki67, by Fortis Life Sciences (1 mentions)
Mouse anti pdgfrα, by Santa Cruz Biotechnology (1 mentions)
C1002, by Beyotime (1 mentions)
13 protocols using p0083
1
Immunohistochemical Analysis of c-Fos in LPBN
2
Immunohistochemical Analysis of Heart Samples
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Heart samples were collected and fixed overnight in 4% paraformaldehyde (BL539A, Biosharp, Hefei, Anhui, China), followed by routine dehydration and sectioning (5 μm). Sections were blocked with 3% hydrogen peroxide, antigen repaired with citrate buffer (P0083, Beyotime, Shanghai, China), permeabilized with 0.3% Triton-100 (ST795, Beyotime, Shanghai, China), blocked with 5% BSA (A1933, Sigma-Aldrich, St. Louis, MO), incubated with primary antibody overnight at 4 °C and incubated with HRP-labeled secondary antibody for 30 min at 37 °C. Visualization was performed under the microscope (DFC700T, Leica, Germany) with DAB Horseradish Peroxidase Color Development Kit (P0203, Beyotime, Shanghai, China). Finally, the sections were sealed with neutral balsam fixative (G8590, Solarbio, Beijing, China). The positive cell number was counted from 4–5 fields per sample with the ImageJ software program (v.1.45, National Institutes of Health, Bethesda, MD, USA) and the mean density was quantified from 4–5 fields per sample with the Image-pro plus software program (v.6.0, Media Cybernetics, Rockville, MD, USA). The antibodies are listed in Supplementary Table 1 .
3
Placental Tissue Histology and Immunohistochemistry
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Placental tissues embedded in wax were sliced at a thickness of 5 μm. For the hematoxylin and eosin (H&E) assay, the slides were stained with hematoxylin for 5 min and eosin Y for 30 s (C0105S, Beyotime, Beijing, China). Images were captured using an Olympus DP71 microscope. For immunohistochemistry assay, antigen retrieval was carried out in citrate buffer pH 6.0 (P0083, Beyotime, Shanghai, China) using a pressure cooker for 3 min, followed by blocking endogenous peroxidases using 0.3% H2O2 for 15 min. After blocking 20 min by blocking buffer (P0260, Beyotime, Shanghai, China), the slides were incubated overnight at 4°C with an anti-CPT2 antibody (1:150, ab181114, Abcam, Cambridge, UK), anti-CPT1b antibody (1:150, 22170-1-AP, Proteintech, Wuhan, China). On the next day, the slides were washed and then incubated with a secondary antibody (PV9001, Zhongshan Gold Bridge Biotechnology Co, Beijing, China) for 20 min at room temperature. The slides were incubated with DAB (ZLI9018, Zhongshan Gold Bridge Biotechnology Co, Beijing, China) to detect side-specific antigen-antibody binding, followed by staining with hematoxylin. Then, the slides were dehydrated and sealed with neutral gum. Images were captured using an Olympus DP71 microscope. Five random views per tissue section were used to quantify the mean IOD (IOD/area) using Image-Pro Plus 6.0.
4
Heart Tissue Immunofluorescence Staining
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Heart samples were collected and fixed in 4% paraformaldehyde (BL539A, Biosharp, Hefei, China) overnight followed by conventional dehydration and slicing (5 μm). The heart samples sections were blocked with 3% hydrogen peroxide and then performed at 95 °C for 10 min using citrate buffer (P0083, Beyotime, Shanghai, China), 0.3% Triton-100 (ST795, Beyotime, Shanghai, China) was used for permeabilization and then the blocking step was carried out using the 5% BSA (A1933, Sigma-Aldrich, St. Louis, MO). After overnight incubation of the primary antibody at 4 °C, a secondary antibody was applied to the sections at 37 °C for 90 min. All the immune-fluorescence images were captured by a fluorescence microscope (DFC700T, Leica, Germany). The antibody is listed in Supplementary Table 1 .
5
Immunostaining of Pdgfr-Alpha and Ki67
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Immunostaining was performed in paraffin sections according to the standard protocols. In brief, samples were deparaffinized and rehydrated, followed by antigen retrieval in 10 mm sodium citrate (Beyotime, P0083). Blocking and staining were performed in antibody diluent with 10% Goat serum. Sections were incubated with mouse anti‐Pdgfrα (Santa Cruz, sc‐398206) and rabbit anti‐Ki67 (Bethyl Laboratories, IHC‐00375) antibodies overnight at 4 °C, followed by incubation with the corresponding secondary antibodies goat anti‐mouse Alexa Fluor 594 (Thermo Fisher, A11032) and anti‐rabbit Alexa Fluor 488 (Thermo Fisher, A11034), respectively. Slides were mounted with DAPI (4′,6‐diamidino‐2‐phenylindole) and imaged by fluorescence microscopy. Cell counting was achieved using the ImageJ software.
6
Immunofluorescence Staining of Paraffin Sections
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After dewaxing and rehydrating according to a previous description, paraffin sections were repaired in citrate solution (P0083, Beyotime, Shanghai, China) for 30 min, blocked with 5% bovine serum albumin (BSA) for 60 min, and incubated with primary antibodies overnight at 4°C. Subsequently, they were incubated with secondary antibodies for 60 min and DAPI (C1002, Beyotime, Shanghai, China) for 15 min, followed by visualization using an immunofluorescence microscope and analyzed with ImageJ software.
7
Myocardial Cell Membrane and TNF-α Staining
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The myocardial cell membrane was stained with WGA antibody (ZF0305, Vector Laboratories), diluted with 5% BSA at a ratio of 1:500, incubated for 2 h at room temperature, washed with PBS 1X, and then mounted with DAPI anti-fluorescence quenching mounts, and collected by a microscope.
The paraffin sections of the heart were deparaffinized. According to the instructions of the reagents, used 1% H2O2 for 10 min, Used sodium citrate repair solution (P0083, Beyotime) to repair antigen in a microwave oven, and 5% BSA for 30 min. Afterward, incubate with TNF-α primary antibody (AF-410-SP, R&D) at 20 ug/ml at 4°C overnight. After washing with PBS 1X, add anti-goat secondary antibody and incubate for 30 min. SABC reacted for 30 min, washed and stained with DAB staining solution, hematoxylin stained the nucleus, and then mounted the slide and took pictures under a microscope.
The paraffin sections of the heart were deparaffinized. According to the instructions of the reagents, used 1% H2O2 for 10 min, Used sodium citrate repair solution (P0083, Beyotime) to repair antigen in a microwave oven, and 5% BSA for 30 min. Afterward, incubate with TNF-α primary antibody (AF-410-SP, R&D) at 20 ug/ml at 4°C overnight. After washing with PBS 1X, add anti-goat secondary antibody and incubate for 30 min. SABC reacted for 30 min, washed and stained with DAB staining solution, hematoxylin stained the nucleus, and then mounted the slide and took pictures under a microscope.
8
Immunohistochemical Analysis of Tumor Markers
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Tumor tissues were fixed in 4% formaldehyde, dehydrated with gradient concentrations ethanol, embedded into parrafin (YA0011, Solarbio) and cut into sections with a thick of 5 μm. Sections were retrieved in 10 mM sodium citrate buffer (pH 6.0, P0083, Beyotime) for 15 min at 94°C. Following cooling to room temperature, sections were blocked with 1% bovine serum albumin (BSA, ST2249, Beyotime) for 30 min and then incubated with primary antibodies against RAD54B (1:200, ab238579, Abcam), Ki-67, c-myc (1:1000, ab32072, Abcam) and MMP-7 (1:500, ab216631, Abcam) respectively. Subsequently, sections were incubated with biotinylation-labeled secondary antibody (1:1000, ab207996, Abcam), re-stained with hematoxylin, and captured under a light microscope (Olympus). The relative level of RAD54B, Ki-67, cmyc and MMP-7 was determined as the ratio of the number of positive cells to the total number of cells.
9
Immunohistochemical Profiling of Murine Tumor Biomarkers
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The biomarkers in nude mice transplanted tumors were detected by IHC methods. Cut the wax block into 4-μm wax slices, and use an oven at 60°C for 1–2 h. The sections were removed for dewaxing, hydration, and then antigen repair with improved sodium citrate antigen repair solution (P0083, Beyotime, China). Incubated for 30 min with inactivated endogenous peroxidase 3% H2O2 (SV0004, BOSTER, China) to avoid light. Afterwards, they were blocked with ready-to-use normal goat serum (AR0009, BOSTER, China) for 30 min. After shaking off the blocking solution, Ki67 (1:300, ab15580, abcam, United Kingdom), HMOX1 (Rabbit, 1:200, 10701-1-AP, proteintech, China), LC3 (Rabbit, 1:500, 14600-1-AP, proteintech, China), CTSL (Rabbit, 1:500, proteintech, China) were added and placed in a refrigerator at 4°C overnight for incubation. Polymeric HRP-labeled anti-rabbit/mouse IgG (SV0004, BOSTER, China) was added after washing with PBS and incubated for 30 min at room temperature in the dark. The color was developed with DAB chromogen solution (C02-12, ORIGENE, America). After color development, counterstain with hematoxylin (AR0005, BOSTER, China). Afterwards, it was blue with alkaline PBS. Finally, dehydration was performed and the slides were mounted. Scanning observations were performed using a Pannoramic Desk (3DHISTECH, Hungary).
10
Immunohistochemistry of Mouse Testis
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Immunohistochemistry was performed on mouse testis tissue sections using standard protocols: after being dewaxed and rehydrated, the paraffin sections were repaired with sodium citrate repair solution (P0083; Beyotime, China). Next, sections were incubated with anti-LC3B (18725-1-AP, Proteintech, USA; 1:200) and anti-F4/80 (70076, Cell Signaling Technology; 1:200) primary antibodies overnight at 4°C. The slices were then treated with biotinylated secondary antibodies and incubated with peroxidase-linked streptavidin (Nakasugi Golden Bridge, China). Hematoxylin was used as a counterstaining agent; adding 3,3'-diaminobenzidine (DAB) allowed the staining to be visualized with a microscope (Nikon, Japan).
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