Primary antibodies used are summarized in Table 1 . Immunostaining was performed as reported previously by our group [13 (link)–15 (link)]. Briefly, serial coronal sections were mounted on 3-aminopropyl triethoxysilane-coated slides. Every eighth section from the habenular to the posterior commissure (8–10 sections per animal) was examined using unbiased stereological principles. The sections for testing Aβ (4G8 antibody) were deparaffinized, hydrated, and pretreated with formic acid (88%) and subsequently with 3% H2O2 in methanol. The sections for testing total tau (HT7 antibody), and phospho-tau epitopes, were deparaffinized, hydrated, subsequently pretreated with 3% H2O2 in methanol, and then treated with citrate (10 mM) or IHC-Tek Epitope Retrieval Solution (IHC World, Woodstock, MD) for antigen retrieval. Sections were blocked in 2% fetal bovine serum before incubation with primary antibody overnight at 4 °C. Next, sections were incubated with biotinylated anti-mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) and then developed by using the avidin-biotin complex method (Vector Laboratories) with 3,3′-diaminobenzidine as a chromogen. Light microscopic images were used to calculate the area occupied by the immunoreactivities by using the software Image-Pro Plus for Windows version 5.0 (Media Cybernetics, Bethesda, MD).
Biotinylated anti mouse immunoglobulin g
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated anti-mouse immunoglobulin G is a laboratory product that binds to mouse immunoglobulin G (IgG) molecules. It is commonly used as a detection reagent in various immunoassay and immunohistochemistry techniques.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin complex method, by Vector Laboratories (5 mentions)
Image pro plus for windows version 5, by Media Cybernetics (4 mentions)
Ihc tek epitope retrieval solution, by IHC World (3 mentions)
Vectastain abc reagent, by Vector Laboratories (1 mentions)
Mouse anti ed1, by Bio-Rad (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Abc elite kit, by Vector Laboratories (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Aperio imagescope software, by Leica (1 mentions)
Goat serum, by Vector Laboratories (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
Aperio scanscope fl, by Leica (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Mouse anti hif 1α, by Santa Cruz Biotechnology (1 mentions)
Avidin biotin peroxidase complex, by Vector Laboratories (1 mentions)
10 protocols using biotinylated anti mouse immunoglobulin g
1
Quantitative Immunohistochemical Analysis of Alzheimer's Pathology
2
Immunohistochemical Analysis of Alzheimer's Pathology
3
Immunohistochemical Analysis of β-Catenin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small intestines were fixed, embedded, and sectioned as Swiss rolls for further immunohistochemical examination with the avidin–biotin complex immunoperoxidase technique and monoclonal mouse anti-β-catenin antibody (Ab; BD Transduction Laboratories, Franklin Lakes, NJ) at 100× dilution. The secondary Ab, biotinylated antimouse immunoglobulin G (Vector Laboratories, California, CA), was used at a 200× dilution. Staining was performed using avidin–biotin reagents (Vectastain ABC reagents; Vector Laboratories), 3,3'-diaminobenzidine and hydrogen peroxide, and the sections were counterstained with hematoxylin to facilitate orientation. As a negative control, consecutive sections were immunostained without exposure to the primary Ab.
4
Immunohistochemical Staining of Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the experiment, the cultures were fixed in 4% paraformaldehyde and permeated using 0.2% Triton X-100 in PBS for 10 min, as previously described [23] (link), [25] (link). The background staining was reduced by blocking nonspecific binding sites with 10% goat serum for 1 h at RT. After rinsing with PBS, the endogenous peroxidase activity was quenched by incubation with 3% H2O2 in PBS for 10 min. The cultures were rinsed again and incubated overnight with appropriate primary antibodies (mouse anti-NeuN, 1:500, Chemicon, Temecula, CA, USA; mouse anti-GFAP, 1:1000, Chemicon; and mouse anti-ED1, 1:500, Serotec, Bicester, UK) at 4 °C. The cells were then rinsed three times with PBS and incubated with a secondary antibody (biotinylated antimouse immunoglobulin G, at 1:200, Vector Laboratories Burlingame, CA, USA) for 1 h. After incubation, the cells were washed with PBS three times (15 min per wash) and visualized using the avidin-biotin peroxidase complex method (ABC Elite kit; Vector Laboratories).
5
Immunohistochemical Analysis of Tau and Synaptic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies used in this study are listed on Table 1 . Immunostaining was performed as reported previously.12 (link), 13 (link) Briefly, serial 6-μm thick coronal sections were mounted on 3-aminopropyl triethoxysilane-coated slides. Every eighth section from the habenular to the posterior commissure (8–10 sections per animal) was examined using unbiased stereological principles. The sections for testing total tau (HT-7), phospho-tau (PHF-1, AT8, AT270), synaptophysin (SYP) and microtubule-associated protein-2 (MAP-2) were deparaffinized, hydrated, subsequently pretreated with 3% H2O2 in methanol and then treated with citrate (10 mm ) for antigen retrieval. Sections were blocked in 2% fetal bovine serum and then incubated with primary antibody overnight at 4 °C. The following day, sections were incubated with biotinylated anti-mouse immunoglobulin G (Vector Laboratories, Burlingame, CA, USA) and then developed by using the avidin–biotin complex method (Vector Laboratories) with 3,3′-diaminobenzidine as a chromogen. Consecutives sections were incubated in the absence of primary antibodies to ensure specificity of staining.
6
Immunohistochemical Analysis of Alzheimer's Pathology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies used in this study are listed in Table 1 . Immunostaining was performed as reported previously.13 , 14 , 15 , 16 Briefly, serial 6‐μm thick coronal sections were mounted on 3‐aminopropyl triethoxysilane‐coated slides. Every eighth section from the habenular to the posterior commissure (8–10 sections per animal) was examined using unbiased stereological principles. The sections for Aβ were deparaffinized, hydrated, pretreated with formic acid (FA; 88%) and subsequently with 3% hydrogen peroxide in methanol. The sections for tau were deparaffinized, hydrated, treated with 3% H2O2 in methanol and citrate (10 mmol/L) for antigen retrieval. Sections were blocked in 2% fetal bovine serum and incubated with primary antibody for total tau (HT‐7), phospho‐tau (PHF‐13, AT8), synaptophysin (SYP), microglia (Iba1) and Aβ (4G8) overnight at 4°C. The following day, sections were incubated with biotinylated anti‐mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) and then developed by using the avidin–biotin complex method (Vector Laboratories) with 3,3′‐diaminobenzidine as a chromogen. Consecutives sections were incubated in the absence of primary antibodies to ensure specificity of staining.
7
Immunohistochemical Detection of IFNε
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HIER was done using a Biocare decloaking chamber in citrate buffer (10 mmol/L trisodium citrate, pH 6.0) at 110°C for 5 minutes under pressure. To detect IFNε protein expression, mouse anti-IFNε (Clone HE70; S. S. Lim, manuscript in preparation) was used at a concentration of 3 μg/mL. Background staining was prevented by using goat serum (Vector Laboratories). Biotinylated anti-mouse immunoglobulin G (Vector Laboratories) was used as secondary antibody, followed by incubation with Alexa Fluor 488-labelled streptavidin (Invitrogen).
Slides were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Molecular Probes) and coverslipped with fluorescence mounting medium (Agilent).
Slides were scanned at 20× and 40× magnification using an Aperio Scanscope FL (Leica Biosystems) and analyzed with Aperio ImageScope software (Leica Biosystems).
Slides were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Molecular Probes) and coverslipped with fluorescence mounting medium (Agilent).
Slides were scanned at 20× and 40× magnification using an Aperio Scanscope FL (Leica Biosystems) and analyzed with Aperio ImageScope software (Leica Biosystems).
8
Quantitative GSAP Immunostaining Analysis
9
Immunohistochemical Analysis of Alzheimer's Pathology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies used are summarized in the Table . Immunostaining was performed as reported previously by our group [16 (link)–18 (link)]. Briefly, serial coronal sections were mounted on 3‐aminopropyl triethoxysilane‐coated slides. Every eighth section from the habenular to the posterior commissure (8–10 sections per animal) was examined using unbiased stereological principles. The sections for testing Aβ (4G8 antibody) were deparaffinized, hydrated, and pretreated with formic acid (88%) and subsequently with 3% H2O2 in methanol. The sections for testing total tau (HT7 antibody), and phospho‐tau epitopes, were deparaffinized, hydrated, subsequently pretreated with 3% H2O2 in methanol, and then treated with citrate (10mM) or IHC‐Tek Epitope Retrieval Solution (IHC World, Woodstock, MD) for antigen retrieval. Sections were blocked in 2% fetal bovine serum before incubation with primary antibody overnight at 4°C. Next, sections were incubated with biotinylated anti‐mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) and then developed by using the avidin‐biotin complex method (Vector Laboratories) with 3,3′‐diaminobenzidine as a chromogen. Light microscopic images were used to calculate the area occupied by the immunoreactivities by using the software Image‐Pro Plus for Windows version 5.0 (Media Cybernetics, Bethesda, MD).
10
Immunohistochemical and Immunofluorescent Analysis of HIF-1α Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical examination was performed to observe spatial profiles of HIF-1α. Briefly, sections were brought to room temperature and incubated in 3% H 2 O 2 for 15 minutes. After washing 3 times in phosphate-buffered saline for 5 minutes each, nonspecific binding was blocked in 5% bovine serum albumin (Sigma Aldrich Co.) at 37°C for 1 hour. Sections were incubated with mouse anti-HIF-1α (1:200, Santa Cruz Biotech), with a biotinylated anti-mouse immunoglobulin G (1:200) for 1 hour, and then with avidin-biotin-peroxidase complex (1:100, Vector Laboratories) at 37°C for 1 hour. Immunoreactivity was visualized with diaminobenzidine (DAB, Boster Biotech Co.).
Immunofluorescence double labeling was used to detect the expression of HIF-1α in ECs. The sections were first incubated for 48 hours at 4°C with a mixture of two primary antibodies against vWF (1:200) and HIF-1α (1:100). The following secondary antibodies were used: Fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody (1:50, Santa Cruz Biotechnology) for vWF detection; rhodamineconjugated donkey anti-mouse antibody (1:100, Santa Cruz Biotechnology) for HIF-1α. These sections were scanned using a laser scanning confocal microscope (LSM-510, Zeiss, Germany).
For the negative control, 1% BSA was used instead of the primary antibody in each experiment.
Immunofluorescence double labeling was used to detect the expression of HIF-1α in ECs. The sections were first incubated for 48 hours at 4°C with a mixture of two primary antibodies against vWF (1:200) and HIF-1α (1:100). The following secondary antibodies were used: Fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody (1:50, Santa Cruz Biotechnology) for vWF detection; rhodamineconjugated donkey anti-mouse antibody (1:100, Santa Cruz Biotechnology) for HIF-1α. These sections were scanned using a laser scanning confocal microscope (LSM-510, Zeiss, Germany).
For the negative control, 1% BSA was used instead of the primary antibody in each experiment.
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!