In short, first, we fixed tissues on glass slides, used xylene and alcohol to deparaffinize and hydrate sections, and washed them with tap water. Second, we blocked endogenous peroxidase activity with 3% H2O2 and preformed antigen retrieval with pressure cooker in 10 mM citrate buffer. Third, we incubated the sections with antibodies against CLDN7 (Sigma, St Louis, MO, USA; 1:100) overnight at 4°C after blocked with nonspecific binding. Last, we used a horseradish peroxidase (HRP)-conjugated secondary antibody (Maixin-Bio, Fujian, China) to bind the primary antibodies and visualized sections with 3,3-diaminobenzidine solution (Maixin-Bio).
Hrp conjugated secondary antibody
Manufactured by Maixin Group
Sourced in China
HRP-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques. It consists of a secondary antibody that is conjugated with the enzyme horseradish peroxidase (HRP). The HRP-conjugated secondary antibody binds to the primary antibody and enables the detection of target analytes through a colorimetric or chemiluminescent reaction.
Automatically generated - may contain errors
4 protocols using hrp conjugated secondary antibody
1
Immunohistochemical Analysis of CLDN7
2
Immunofluorescent and Immunohistochemical Analysis of Mast Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescent staining, sections of lungs or cells were incubated with the antibody against mast cell tryptase (ab2378, Abcam, UK), Tespa1 (R1309-16, HuaAn Biotechnology, China), p-STAT6 (ab263947, Abcam, UK) and DAPI (4ʹ,6-diamidino-2-phenylindole, Life Technologies,), and images were obtained by using a confocal laser scanning microscope (LSM 880, Zeiss). The protein expression levels were analyzed using Image J.1.44 software.
For immunohistochemical staining, the slides were incubated with 3% H2O2 for 10 min after dewaxing, and then washed with PBS for 5 min at room temperature. Antigen retrieval was performed in citrate buffer (pH 6.0) by microwave heating, and blocking was performed with 10% non-immune goat serum for 30 min after cooling. The slides were incubated with an antibody against mast cell tryptase (ab2378, Abcam, UK) overnight at 4 ℃. After rinsing with PBS, the sections were incubated with the HRP-conjugated secondary antibody (Maixin, Fuzhou, China) for 30 min at room temperature. Hematoxylin was applied as a counterstain. Eight fields were randomly selected for the quantification of positive cells in every sample, as previously described [19 (link)].
For immunohistochemical staining, the slides were incubated with 3% H2O2 for 10 min after dewaxing, and then washed with PBS for 5 min at room temperature. Antigen retrieval was performed in citrate buffer (pH 6.0) by microwave heating, and blocking was performed with 10% non-immune goat serum for 30 min after cooling. The slides were incubated with an antibody against mast cell tryptase (ab2378, Abcam, UK) overnight at 4 ℃. After rinsing with PBS, the sections were incubated with the HRP-conjugated secondary antibody (Maixin, Fuzhou, China) for 30 min at room temperature. Hematoxylin was applied as a counterstain. Eight fields were randomly selected for the quantification of positive cells in every sample, as previously described [19 (link)].
3
Immunohistochemical Analysis of Colon Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry (IHC) was used to detect the expression of TNF-α, IL-1β, IL-6, occludin, ZO-1 and MUC2. Briefly, 4-μm-thick colon tissue slides were subjected to heat-induced antigen retrieval after dewaxing and hydration. After antigen retrieval, the sections were incubated with 3% H2O2 solution at room temperature for 15 min for blocking endogenous peroxidase activity. After the sections were blocked with 10% normal goat serum for 60 min, the tissues were incubated with rabbit polyclonal antibodies for TNF-α (1:200), IL-1β (1:50), IL-6 (1:50), occludin (1:100), ZO-1 (1:100), or MUC2 (1:500) overnight at 4°C, followed by HRP-conjugated secondary antibody (Maixin, Fuzhou, China) for 60 min at room temperature. A diaminobenzidine (DAB) kit (Maixin; Cat No. DAB-2031) was used for color development and the sections were counterstained with hematoxylin. The tissues on each slide were imaged under a light microscope (Leica, Germany) at ×400 magnification. And the percentage of positive cells from 6 fields was determined using ImageJ (open-source Java image processing program, https://imagej.nih.gov/ij/ ).
4
Immunohistochemical and Immunofluorescence Staining of Corneal Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemical staining, the corneas were fixed in 4% paraformaldehyde, embedded in paraffin and then sectioned. After treatment with 3% H2O2, corneal sections were incubated with primary antibodies overnight at 4 °C and stained with an HRP-conjugated secondary antibody for 1 h at 37 °C (MaiXin Biotechnology, Fuzhou, China). All staining was examined under a Nikon fluorescence microscope. Antibodies used for immunohistochemical staining: rabbit anti-Cathepsin D (1:1000, Abcam ab75852), rabbit anti-Cathepsin K (1:1000, Abcam ab207086), rabbit anti-KRT80 (1:1000, Proteintech 16835-1-AP), rabbit anti-YAP1 (1:1000, Proteintech 13584-1-AP), rabbit anti-SPRR1B (1:1000, Proteintech 11959-1-AP).
For immunofluorescence staining, corneal tissues were fixed in 4% paraformaldehyde and blocked with 5% normal serum for 30 min at room temperature. The samples were treated with primary antibodies overnight at 4 °C and subsequently with secondary antibodies for 1 h at 37 °C. Nuclei were stained with 4',6 diamidino-2-phenylindole (DAPI). The flat mounts were examined and captured under a confocal microscopy (LSM800, Zeiss, Germany). Antibodies used for immunofluorescence staining: rabbit anti-K10 (1:1000, Abcam ab76318), rabbit anti-K12 (1:1000, Abcam ab185627), rabbit anti-PAX6 (1:1000, Proteintech 12323-1-AP).
For immunofluorescence staining, corneal tissues were fixed in 4% paraformaldehyde and blocked with 5% normal serum for 30 min at room temperature. The samples were treated with primary antibodies overnight at 4 °C and subsequently with secondary antibodies for 1 h at 37 °C. Nuclei were stained with 4',6 diamidino-2-phenylindole (DAPI). The flat mounts were examined and captured under a confocal microscopy (LSM800, Zeiss, Germany). Antibodies used for immunofluorescence staining: rabbit anti-K10 (1:1000, Abcam ab76318), rabbit anti-K12 (1:1000, Abcam ab185627), rabbit anti-PAX6 (1:1000, Proteintech 12323-1-AP).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!