The immunohistochemistry was performed as previously described [16 (link)–18 (link)]. Briefly, the sections were first deparaffinized and rehydrated, and the endogenous peroxidase activity was blocked. Sections were then subjected to microwave antigenic retrieval, and the non-specific binding was blocked. Finally, sections were incubated with anti-NEURL3 antibody (1:200, 16648-1-AP; Proteintech) overnight at 4 °C, followed by biotinylated secondary antibody (Boster, Wuhan, China). All of the sections were scored by two pathologists according to the immunoreactive score (IRS) system as previously described [16 (link)–18 (link)].
Biotinylated secondary antibody
Manufactured by Boster Bio
Sourced in China
Biotinylated secondary antibodies are laboratory reagents used to detect and amplify the signal of target primary antibodies in various immunoassay techniques. They bind to the primary antibody and provide a platform for further signal enhancement through the high-affinity interaction between biotin and streptavidin.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Boster Bio (2 mentions)
Hematoxylin, by Wuhan Servicebio Technology (1 mentions)
Avidin biotin peroxidase, by Boster Bio (1 mentions)
Eclipse 80i microscope, by Olympus (1 mentions)
Anti phospho erk antibody, by Cell Signaling Technology (1 mentions)
Diaminobenzidine substrate kit, by Boster Bio (1 mentions)
Ab38898, by Abcam (1 mentions)
Ab1316, by Abcam (1 mentions)
Primary antibody against α sma, by Abcam (1 mentions)
Biotinylated habp, by Merck Group (1 mentions)
Anti cd34 antibody, by Abcam (1 mentions)
Sc 79, by Santa Cruz Biotechnology (1 mentions)
Inverted fluorescence microscope, by Leica (1 mentions)
Alox15, by Abcam (1 mentions)
Dab kit, by Boster Bio (1 mentions)
Slc7a11, by Proteintech (1 mentions)
Acsl4, by Santa Cruz Biotechnology (1 mentions)
Strept avidin biotin complex, by Boster Bio (1 mentions)
Bovine serum albumin (bsa), by Boster Bio (1 mentions)
18 protocols using biotinylated secondary antibody
1
Immunohistochemical Analysis of NEURL3 Expression
2
Immunohistochemical Detection of FHL2 in Ovarian Tissues
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A previously described peroxidase-based immunohistochemistry protocol was used to detect FHL2 expression in paraffin-embedded ovarian tissues [36 (link)]. Anti-FHL2 antibody (Proteintech, Wuhan, China) was used at 1:200. Subsequently, the sections were incubated with biotinylated secondary antibody (1:2000) (Boster Biotechnology, Wuhan, China) and avidin-biotin-peroxidase (Boster Biotechnology, Wuhan, China) before being exposed to diaminobenzidine (Servicebio, Wuhan, China, Wuhan, China) and counterstained with hematoxylin (Servicebio, Wuhan, China).
3
Immunohistochemical Analysis of NMDA Receptors
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The L4-L5 spinal cord segments were extracted rapidly after the last behavioral test. 4% paraformaldehyde in phosphate buffered saline (PBS) was applied to fix the samples for 6 h, and then paraffin was used to embed the samples. The samples were cut into 7-μm-thick sections and placed onto glass slides. After deparaffinization and rehydration in descending concentrations of ethanol, the sections were treated with avidin-biotin peroxidase for 15 min. To make the results more intuitive, citrate solution was used for antigen retrieval. 5% normal goat serum was selected for treatment to block non-specific reactions for 1h at room temperature. Then the sections were incubated with primary antibody of rabbit polyclonal anti-NR1, NR2A, NR2B (1:200; Abcam, Cambridge, UK) overnight at 4°C respectively. After rewarming for 45min, the sections were incubated with biotinylated secondary antibody (1:300, Boster Biological Technology, Ltd., Wuhan, China) for 30 min at room temperature and stained by diaminobenzidine (DAB substrate kit, Boster Biological Technology). Sections were counterstained with hematoxylin, differentiated by ethanol hydrochloride, dehydrated in ascending series of ethanol, cleared in xylene and coverslipped with neutral gum. Images were obtained from an Olympus eclipse 80i microscope (Olympus, Tokyo, Japan).
4
HIF-1α Protein Expression Analysis
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The expression of HIF-lα protein was detected by streptavidin-peroxidase conjugate as previously described [13 (link)]. Heart and lung tissues were fixed, sliced, and inactivated. Then, sections were blocked and incubated with the primary rabbit anti-HIF-1α polyclonal antibody (1: 200, Boster Biological Technology Co.) at 4°C overnight and with the biotinylated secondary antibody (1: 100, Boster) for 1 h. After added horseradish peroxidase-tagged streptavidin working solution (S-A/HRP), the sections were developed in DAB (Boster Biological Technology Co.). The mean absorbance (A) of pulmonary arterial wall was detected and used as the relative protein expression levels of HIF-lα.
5
Cartilage Tissue Analysis in UAC Rats
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Rat mandibular condyle cartilage tissues (n = 24, CON: UAC = 12:12) were fixed with 4% paraformaldehyde, decalcified with buffered EDTA (12.5% EDTA, pH 7.4), and embedded in paraffin. The paraffin specimens were cut into 5-μm thick sections. The tissue sections were deparaffinized in xylene solution and rehydrated in a graded series of ethanol solutions. Sections of damaged cartilage from UAC rats and normal cartilage were stained with Safranin O and counterstained with Fast Green. For IHC, after a 3% H2O2 treatment and antigen retrieval, all tissue sections were blocked at room temperature with 5% bovine serum albumin for 30 min and then incubated with the following primary antibodies at the indicated dilutions: anti-ERRγ antibody (mouse; sc-393969; 1:100; Santa Cruz); anti–phospho-ERK antibody (rabbit; #4370; 1:250; Cell Signaling Technology); anti-MMP9 antibody (rabbit; ab38898; 1:100; Abcam); and anti-VEGFA antibody (rabbit; ab1316; 1:100; Abcam). After sequential incubations with a biotinylated secondary antibody (BOSTER, SA1022, China) and horseradish peroxidase-conjugated avidin (BOSTER, SA1022, China), proteins were labeled with a Diaminobenzidine Substrate kit (BOSTER, SA2022, China).
6
Immunohistochemical Detection of α-SMA
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The sections were dewaxed in xylene and dehydrated in alcohol. Antigen retrieval was achieved by microwaving in citric saline at 95 °C for 3 min. Thin sections were treated with 3 % hydrogen peroxide for 10 min. The sections were further blocked by 5 % BSA and were then incubated 37 °C with primary antibody against α-SMA (Abcam, UK). The sections were incubated with biotinylated secondary antibody (Boster, Wuhan, China) for 30 min at room temperature. α-SMA expressions were visualized by DAB (Boster, Wuhan, China) staining.
7
Immunohistochemical Analysis of HA and CD204 in Breast Cancer
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Immunohistochemical staining of HA and CD204 were performed using the formalin-fixed, paraffin-embedded biopsy specimens of breast cancer and breast benign disease. Briefly, 5-μm sections were deparaffinized, rehydrated and microwaved in sodium citrate for antigen-retrieval. The sections were blocked at room temperature for 1 h with 5% BSA in PBS. For HA staining, the slides were incubated with biotinylated HABP (Merck, Germany, dilution: 1:50) at 4ºC overnight. Slides were then washed three times with PBS and incubated with Streptavidin-ABC for 30 min at room temperature. To detect CD204 expression, the slides were incubated with anti-human CD204 antibodies (Molecular Probes, USA, dilution: 1:100) overnight at 4ºC. Next day, the slides were rinsed with PBS before incubation with biotinylated secondary antibody (Boster, China) for 30 min at room temperature. After removing non-reacted secondary antibodies, the sections were incubated with Streptavidin-ABC and then developed with DAB Substrate Kit to visualize the immunolabeling.
8
Immunohistochemical Analysis of Nogo-B and CD34
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Paraffin‐embedded specimens were cut into 5‐μm‐thick sections, deparaffinized, and rehydrated through a decreasing ethanol gradient. Endogenous peroxidase was first blocked with H2O2. After BSA blocking, slides were incubated with anti‐Nogo‐B (1 : 200 dilution; Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or anti‐CD34 antibody (1 : 100 dilution; Abcam, Cambridge, UK), which was followed by incubation with biotinylated secondary antibody (1 : 100 dilution; Boster, Wuhan, China). The presence of the avidin–biotin complex was finally revealed with diaminobenzidine. Quantitative analysis of the Nogo‐B intensity, CD34‐positive blood vessel density, and blood vessel area was performed using imagej software.
9
Immunohistochemical Staining Protocol
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In short, paraffin embedded sections are cut, deparaffined, rehydrated, and then heat treated for antigen recovery. Next, the slices were incubated with 3% hydrogen peroxide to inactivate endogenous peroxidase activity, and then incubated with 5% normal blocking serum. After that, the sections were treated with specific primary antibody overnight, then biotinylated secondary antibody (boster, Wuhan, China) and SABC solution. Finally, the sections were observed and imaged by light microscope.
IHC staining intensity (0, negative; 1, Weak; 2, Medium; 3, strong) and immunostaining area (0, 0–5%; 1, 6–25%; 2, 26–50%). If the total score is less than 3, it is negative. If the total score is more than 3, it is positive.
IHC staining intensity (0, negative; 1, Weak; 2, Medium; 3, strong) and immunostaining area (0, 0–5%; 1, 6–25%; 2, 26–50%). If the total score is less than 3, it is negative. If the total score is more than 3, it is positive.
10
Neurotrophin-3 Expression Analysis
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After the experiment, rats were anesthetized by a 10% chloral hydrate solution, and then secured on an animal operating table. A perfusion cannula was inserted into the aorta through which 250 mL of saline solution was perfused quickly, followed by 2000 mL of phosphate-buffered saline perfused slowly over 1–2 h. Rats were killed rapidly by decapitation and the bilateral cochlear nucleus and inferior colliculus were removed [12 ]. After paraffin embedding, antigen retrieval, and blocking, sections were incubated overnight at 4°C with a rabbit anti-rat NT3 antibody (Boster Biological Technology, China). After washing with PBS, the sections were incubated with a biotinylated secondary antibody (Boster Biological Technology, China) for 30 min at room temperature. Immunoreactivity was visualized by 3,3-diaminobenzidine (DAB). Sections were washed thoroughly, mounted onto gelatin-coated slides, dehydrated, and cover-slipped before being examined under a microscope operated in the bright field mode. Images (40× magnification) from 5 non-overlapping horizons were taken for each slice. The average optical density value was measured by Image-pro Plus (version 6.0).
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