Formalin-fixed paraffin-embedded slides of human pulmonary GVHD were dewaxed with xylene, hydrated with ethanol, incubated with antigen retrieval buffer (Agilent, Santa Clara, CA), and blocked with 3% bovine serum albumin. Primary antibodies, including anti-human CD4 (EPR6855), CD8a (RPA-T8), CD20 (H1), CD68 (KP1), pAKT S473 (D9E), and pERK1/2 (D1314.4E) (Fluidigm, South San Francisco, CA), were applied overnight at 4°C. Secondary incubation was with Cell ID intercalator-Iridium (Fluidigm), for 30 minutes at room temperature. Stained tissue samples were interrogated by using a Hyperion Imaging System (Fluidigm), and acquired data were visualized by using the MCD viewer (Fluidigm).
Antigen retrieval buffer
Manufactured by Agilent Technologies
Sourced in United States, Denmark
Antigen retrieval buffer is a laboratory reagent used to prepare tissue samples for immunohistochemical analysis. Its core function is to help unmask or expose target antigens within the tissue, which is necessary for the subsequent binding and detection of specific antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (2 mentions)
Cri pannoramic scan whole slide scanner, by 3DHISTECH (2 mentions)
Envision kit, by Agilent Technologies (2 mentions)
Pannoramic viewer, by 3DHISTECH (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Mach2 rabbit ap polymer, by Biocare Medical (2 mentions)
Vulcan fast red chromogen, by Biocare Medical (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Cell id iridium intercalator, by Standard BioTools (1 mentions)
Mcd viewer, by Standard BioTools (1 mentions)
Hyperion imaging system, by Standard BioTools (1 mentions)
Anti foxo1 antibody, by Abcam (1 mentions)
Dako envision dual link system hrp, by Agilent Technologies (1 mentions)
Autostainer plus, by Agilent Technologies (1 mentions)
Ab97049, by Abcam (1 mentions)
Dab substrate kit, by Abcam (1 mentions)
Streptavidin peroxidase conjugate, by Merck Group (1 mentions)
Hematoxylin gill 1, by Merck Group (1 mentions)
34 protocols using antigen retrieval buffer
1
Multiparametric Analysis of Pulmonary GVHD
2
Quantitative Analysis of Spinal Neuron Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spinal cords were removed and fixed as described above.
The sections were deparaffinized, rehydrated, placed in antigen retrieval buffer (Agilent Dako, Santa Clara, CA) for 30 minutes at 95 C, and then allowed to cool for 30 minutes at room temperature. The slides were washed with PBS, blocked with 10% normal donkey serum for 60 minutes, and incubated at 4 C with a monoclonal rabbit anti-mouse NeuN antibody solution (1:150 dilution in PBS containing 1% bovine serum albumin). The slides were washed with PBS and incubated for 1 hour with Cy3-conjugated matched immunoglobulin G (Jackson Laboratories, Inc, West Grove, PA) at 1:150 dilution with PBS containing 1% bovine serum albumin. The nucleus was counterstained with 4 0 , 6-diamidino-2phenylindole (imaged on the blue channel), and Alexa 488-tagged wheat germ agglutinin (imaged on green channel) was used to stain the cell membranes. Microscopic observation and photography were performed with the Leica CTR5500 digital microscope (Leica Microsystems GmbH, Wetzlar, Germany). For quantitative analysis of neuronal viability, 3 sections from 1 mouse were collected blindly, and the number of NeuN expression in the spinal ventral horn was counted by a blind observer.
The sections were deparaffinized, rehydrated, placed in antigen retrieval buffer (Agilent Dako, Santa Clara, CA) for 30 minutes at 95 C, and then allowed to cool for 30 minutes at room temperature. The slides were washed with PBS, blocked with 10% normal donkey serum for 60 minutes, and incubated at 4 C with a monoclonal rabbit anti-mouse NeuN antibody solution (1:150 dilution in PBS containing 1% bovine serum albumin). The slides were washed with PBS and incubated for 1 hour with Cy3-conjugated matched immunoglobulin G (Jackson Laboratories, Inc, West Grove, PA) at 1:150 dilution with PBS containing 1% bovine serum albumin. The nucleus was counterstained with 4 0 , 6-diamidino-2phenylindole (imaged on the blue channel), and Alexa 488-tagged wheat germ agglutinin (imaged on green channel) was used to stain the cell membranes. Microscopic observation and photography were performed with the Leica CTR5500 digital microscope (Leica Microsystems GmbH, Wetzlar, Germany). For quantitative analysis of neuronal viability, 3 sections from 1 mouse were collected blindly, and the number of NeuN expression in the spinal ventral horn was counted by a blind observer.
3
Immunohistochemistry for FOXO1 and PAX3 in FFPE Tissue
4
Granzyme B Quantification in FFPE Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FFPE tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Antigen retrieval was performed using antigen-retrieval buffer (Dako, Glostrup, Denmark), heated in a steamer. Tissue sections were incubated with a primary antibody directed against granzyme B (EPR8260, 1:100; Abcam, Cambridge, UK). For immunodetection, tissue specimens were incubated with a biotinylated secondary goat anti-rabbit IgG antibody (ab97049, 1:200; Abcam) and streptavidin–peroxidase conjugate (Merck/Sigma-Aldrich, Taufkirchen, Germany). Staining was detected using the DAB Substrate Kit (Abcam), and nuclei were visualized with Hematoxylin Gill I (Sigma-Aldrich, St. Louis, MO, USA) before mounting. The numbers of granzyme B-positive cells per 20× microscopic field were counted manually. At least three 20× microscopic fields were counted per sample. The 20× fields were randomly chosen and did not correspond to the ROIs selected for DSP.
5
Protein Extraction from FFPE Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction from archival FFPE tissue was performed as previously described [13 (link)]. Briefly, two 10 μm sections of each specimen were lysed with an extraction buffer [1x high pH Antigen retrieval buffer (DAKO), 1% NaN3, 1% SDS, 10% glycerol, and protease inhibitor (1 tablet/25 ml, Roche, Basel, Switzerland)]. Afterwards, incubation for 15 mins at 115°C within a pressure cooker followed by microcentrifugation at 13000 rpm for 30 mins at 4°C. Protein was stored -20°C until further use. Protein concentrations were determined using BCA protein assay (Pierce Biotechnology, Rockford, IL) and used according to the manufacturer’s instructions.
6
Immunostaining of DU-145 Xenograft Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed paraffin-embedded DU-145 xenograft tissue sections were deparaffinized, rehydrated and subject to heat-induced antigen retrieval using antigen retrieval buffer (pH 6.0; Dako, Carpinteria, CA) in a pressure cooker. Sections were blocked with 2.5% normal horse serum for 30 min and incubated with mixture of rabbit monoclonal anti-CD133 antibodies (clone D2V8Q, diluted 1:100; Cell signaling, Danvers, MA) and mouse monoclonal anti-TRA (clone TRA-1-60 (S), diluted 1:1000; Cell signaling) or mixture of rabbit monoclonal anti-CD44 antibodies (clone E7K2Y, diluted 1:1000; Cell signaling) and mouse monoclonal anti-TRA (clone TRA-1-60 (S), diluted 1:1000; Cell signaling) for 1 h at room temperature, followed by incubation with mixture of Alexa Fluor 488 goat anti-rabbit IgG (ThermoFisher Scientific, Waltham, MA) and Alexa Fluor 555 goat anti-rabbit IgG (ThermoFisher Scientific). After nucleus visualization with DAPI, sections were mounted with Prolong Diamond antifade (ThermoFisher Scientific) and imaged on a Zeiss Axio Imager 2 (Carl Zeiss Microscopy, Jena, Germany).
7
Immunohistochemical Analysis of Alzheimer's Disease Brain Samples
8
Hematoxylin-Eosin and Immunofluorescence Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Hematoxylin and eosin staining, paraffin sections were dewaxed using xylene for 30 min, hydrated in 100%-, 90%-, 80%- and 70% EtOH and was dipped into Mayer’s hematoxylin (Sigma-Aldrich) for 8 min, and then rinsed in water for 1 minute. Slide was dipped again into eosin Y (Sigma-Aldrich) for 80 s, dehydrated with 70%-, 80%-, 90%- and 100% EtOH, washed with fresh xylene for 30, dried, and mounted with mounting medium. Immunofluorescence staining was performed using standard protocols. Briefly, paraffin sections were dewaxed using xylene for 30 min, hydrated in 100%-, 90%-, 80%- and 70% EtOH and antigen retrieval was performed by boiling using microwave in antigen retrieval buffer (Dako, Carpinteria, CA, USA) for 2 min. The sections were treated with rabbit Ki67 antibody (1:300) (abcam) overnight at 4 °C, and were then incubated with secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen), for 1 h at room temperature with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Immunofluorescence staining was imaged using ZEISS LSM700 confocal microscope.
9
Immunohistochemical Staining of Melanoma Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tks4 and Tks5 antibodies were used to stain melanoma tissue microarray samples obtained from the Cancer Diagnosis Program (CDP) of the National Cancer Institute following approval from the National Disease Research Interchange (NDRI) (http://ndriresource.org/ ). Antigen retrieval was performed according to the manufacturer's guidelines using Antigen Retrieval Buffer (DAKO S1699). After incubation with Tks4 or Tks5 antibody, the sections were incubated with secondary antibody using the manufacturer's guidelines of the MACH2 Rabbit AP-Polymer (Biocare Medical) and visualized with either Vulcan Fast Red Chromogen (Biocare Medical, pink) or DAB (brown). Sections were counterstained with hematoxylin. A scoring system, developed by a trained pathologist (SI), of 0, no staining; 1+, low staining or staining in < 25% of tumor cells (weak positive staining); 2+, moderate staining (positive staining); and 3+, high staining (strong positive staining). All slides were scored blindly.
10
Immunohistochemical Analysis of Tks5 in Breast Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Tks5 antibody (ProteinTech) was first evaluated for specificity and optimal titer by using it to stain paraffin embedded 293 cells transfected with empty vector, or vectors for the adaptor proteins Tks4 and Tks5. The antibody was then used to stain a breast cancer progression tissue microarray obtained from the Cancer Diagnosis Program (CDP) of the National Cancer Institute following approval from the National Disease Research Interchange (NDRI) (http://ndriresource.org/ ). Antigen retrieval was performed according to the manufacturer’s guidelines using Antigen Retrieval Buffer (DAKO S1699). After incubation with the Tks5 antibody, the sections were incubated with secondary antibody using the manufacturer’s guidelines of the MACH2 Rabbit AP-Polymer (Biocare Medical) and visualized with either Vulcan Fast Red Chromogen (Biocare Medical) (pink) or DAB (brown). Sections were counterstained with hematoxylin. A scoring system was developed of 0, no staining; 1+, low staining or staining in <25% of tumor cells; 2+, moderate staining; and 3+, high staining. Slides were independently evaluated by a trained pathologist (S.I.).
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!