Tissue specimens from rat hind limbs and clinical samples were fixed in 4% paraformaldehyde for 48 h and washed with phosphate buffered saline, then dehydrated in a graded ethanol series, vitrified with dimethylbenzene, and inserted in paraffin. Paraffin sections (4 μm) were deparaffinized in xylene, hydrated with gradient ethanol, and stained with standard H&E, SOFG, or Masson staining procedures. To perform the immunohistochemical staining, tissue slides were deparaffinized and rehydrated followed by antigen retrieval, endogenous peroxidase blocking and serum sealing. Then, the slides were incubated with antibodies against UBA1(1:100, ab180125, Abcam Inc, Cambridge, UK), EIF3E (1:50, ab134958, Abcam Inc, Cambridge, UK), RPL27 (1:50, 14980-1-AP, Proteintech, Chicago, USA), RPL17 (1:300, 67223-1-Ig, Proteintech, Chicago, USA), RPS28(1:50, 14796-1-AP, Proteintech, Chicago, USA) at 4 °C overnight. The next day, all sections were taken out and washed with PBS several times. Then the biotinylated secondary antibody (Servicebio, Wuhan, China) was used for 1 h at room temperature followed by the reaction with diaminobenzidine (Servicebio, Wuhan, China) and hematoxylin to develop color. Histological scores were calculated from the results of staining using Image J 6.0 (Media Cybernetics Corporation, USA) software.
Biotinylated secondary antibody
Manufactured by Wuhan Servicebio Technology
Sourced in China
Biotinylated secondary antibody is a labeling reagent that binds to a primary antibody, allowing for signal amplification and detection in various immunological assays. It consists of a secondary antibody conjugated with biotin, a small molecule that can be recognized by streptavidin or avidin. This reagent is commonly used in techniques such as Western blotting, ELISA, and immunohistochemistry to enhance the sensitivity of target protein detection.
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Lab products found in correlation
Diaminobenzidine, by Wuhan Servicebio Technology (1 mentions)
Ab180125, by Abcam (1 mentions)
Ab134958, by Abcam (1 mentions)
Imagej 6, by Media Cybernetics (1 mentions)
Anti ccasp3, by Cell Signaling Technology (1 mentions)
Leica dm4000, by Leica Microsystems (1 mentions)
Streptavidin hrp, by ZSGB-BIO (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Ar1022, by Boster Bio (1 mentions)
Ar0005, by Boster Bio (1 mentions)
Ab220210, by Abcam (1 mentions)
Nb600 1131, by Novus Biologicals (1 mentions)
Optical microscope, by Leica Microsystems (1 mentions)
Citrate buffer, by Beyotime (1 mentions)
Aperio at2 scanner, by Leica Microsystems (1 mentions)
Wgar1009 5, by Wuhan Servicebio Technology (1 mentions)
Gsh px, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
8 protocols using biotinylated secondary antibody
1
Immunohistochemical Profiling of Ribosomal Proteins in Rat and Clinical Samples
2
Immunohistochemical Detection of Cleaved Caspase-3
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After 1 h 0.25% trypsin antigen retrieval and 1 h 10% FBS incubation at room temperature, the sections were incubated with the anti-cCasp3 (1:200, Cell Signaling Technology) primary antibody overnight at 4°C. After that, the sections were incubated with a biotinylated secondary antibody (Servicebio, Wuhan, China) according to the manufacturer's instructions. The sections were stained with a 3,3-diaminobenzidine precipitate and counterstained with hematoxylin. Photomicrographs were acquired using a LEICA DM 4000 (Leica Microsystems, Germany).
3
Immunohistochemical Analysis of Stathmin in Mouse Skin Wound Tissues
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The mouse skin tissues were cut at wound edges and fixed in 4% paraformaldehyde (Cat# P0099, Beyotime, China), embedded in paraffin, and sectioned. Sectioned wound tissues were deparaffinized and rehydrated. Antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0) in a microwave at 600 W for 8 min. To perform immunohistochemistry staining for stathmin, wound tissues were incubated with stathmin rabbit mAb (1:100, Cat# 13655S, Cell Signaling Technology, United States) as the primary antibody at 4°C overnight. The tissues were then rinsed and incubated with a biotinylated secondary antibody (1:500, Cat# GB23303, Servicebio, China) and streptavidin-HRP (Cat#SP-9001, ZSGB-BIO, China). The color was developed using DAB peroxidase substrate (Cat# ZLI-9018, ZSGB-BIO, China) until an optimal color was observed.
4
Immunohistochemical Analysis of Liver Fibrosis
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For IHC analysis, the paraffin-embedded liver sections (4 μm) were pretreated with dewaxed, rehydrated and heat-induced antigen retrieval. Then, the sections were immersed in hydrogen peroxide (3%) for 10 min to neutralize endogenous peroxidase activities and sequentially incubated with goat serum (5%) for 30 min to block non-specific binding sites of the tissues. Next, after immunostaining overnight at 4°C with antibodies against collagen I (1:500; Cat. no. GB11022-1), α-SMA (1:500; Cat. no. GB13044), the slides were immunostained with a biotinylated secondary antibody (1:100; Cat. no. G1216) for 1 h at room temperature. The primary antibodies and biotinylated secondary antibody used in IHC staining were purchased from Wuhan Servicebio Technology Co., Ltd (Wuhan, China). DAB chromogenic reagent (AR1022; Boster; Wuhan, China) was added to visualize positive staining and hematoxylin (AR0005; Boster; Wuhan, China) was used to mark the nucleus. For semi-quantitative analysis of positive cells, images (20× magnification) were acquired by microscopy and analysed using ImageJ V1.8.0 software (National Institutes of Health).
5
Immunohistochemical Analysis of Inflammatory Markers
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After deparaffinization and rehydration, the 4-µm sections were treated with antigen retrieval solution, 0.25% trypsin, at 37 ℃, 30 min and then incubated with 3% H2O2 for 30 min. The samples were blocked with 1% bovine serum albumin (Servicebio, Wuhan, China) in phosphate buffered saline (PBS, 1 × Servicebio, Wuhan, China) for 30 min at room temperature and were incubated with the following primary antibodies overnight at 4 °C: TNF-α (Abcam, ab220210; Species: Rat, Human; Host: Mouse; England) and IL-6 (Novusbio, NB600-1131; Species: Human, Mouse, Rat, Monkey; Host: Rabbit; USA). Slides then were washed three times in PBS and incubated with a biotinylated secondary antibody (Servicebio, Wuhan, China) for 1 h at room temperature. After being rinsed in PBS, the slides were incubated with the avidin-biotin-peroxidase complex for 20 min. Twenty microliters of 50 × DAB stock solution was added to each 1 mL of DAB dilution solution for later use as a chromogen. The slides were counterstained with hematoxylin and rinsed under running water. Image analysis was performed by one skilled evaluator through an optical microscope (Leica Microsystems, Wetzlar, Germany).
6
Histological Analysis of Limb Development
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For histology, limbs were xed in 10% formalin (Servicebio, Wuhan, China) for 24h. For 8-week-old samples, 4-weeks' decalci cation by 10% tetrasodium EDTA were conducted. After dehydrated by graded ethanol and cleared in xylene, limbs were embedded in para n and sectioned at 4μm thickness along the long axis. Sections were stained with safranin O/fast green, masson, hematoxylin and eosin (H&E) (Servicebio, Wuhan, China). For immunohistochemical staining, the antigen retrieval was performed in a citrate buffer (Beyotime, Shanghai, China). Endogenous peroxidase activities were quenched with 3% H 2 O 2 for 10 minutes. Sections were blocked in normal goat serum (NGS) for 1 hour before incubation with primary antibodies overnight at 4°C. The next day, sections were incubated with biotinylated secondary antibody (Servicebio, Wuhan, China) for 1 hour at room temperature. Then, sections were incubated with diaminobenzidine and counterstained with hematoxylin. Phosphate buffer saline (PBS), instead of primary antibody, was used as the negative control. All sections were mounted in neutral resins and then scanned by AperioAT2 scanner (Leica Microsystems).
7
Immunohistochemical Analysis of Oxidative Stress
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After deparaffinization, antigen retrieval was performed by microwaving the sections with citrate buffer (pH 6.0). Endogenous peroxidase activity was inhibited using 3% H2O2 for 15 min, and non-specific binding was blocked with 10% normal goat serum (WGAR1009-5, Servicebio). All sections were incubated with primary antibodies against SOD (1:500, Santa Cruz), GSH-Px (1:500, Abcam), and MDA (5 μg/mL, Abcam) overnight at 4° C. Biotinylated secondary antibodies (1:500, Servicebio) were added and incubated with the sections for 60 min. Their presence was visualized using a diaminobenzidine (DAB) reaction. Finally, the slides were counterstained with hematoxylin. Image-Pro Plus 6.0 (Media Cybernetics, Inc., MD, USA) was used to measure the integrated optical density (IOD) and area of staining.
8
Immunohistochemical Analysis of Intestinal Proteins
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Paraffin-embedded intestinal tissue sections were cut to a thickness of 4 μm, deparaffinized in xylene, and rehydrated in graded alcohols. Antigen retrieval was achieved by microwaving at 100 °C with an antigen unmasking solution (10 mM citrate buffer, pH 6.0). Hydrogen peroxide (3%) was used to eliminate endogenous peroxidase. After serum blocking, the sections were subsequently incubated with primary antibodies against cyclin E, CDK, RAGE (Abcam), CD36 (Novus), and p27kip1 (Servicebio, Wuhan, China) overnight at 4 °C followed by biotinylated secondary antibodies (Zhongshanjinqiao, Beijing, China). Proteins were visualized as brown pigments via a standard diaminobenzidine (Zhongshanjinqiao) protocol. Slides were then lightly counterstained with hematoxylin and positive immunostaining was examined with an Olympus BX-53 microscope (Olympus).
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