Slides were deparaffinized and rehydrated as previously described (Liou et al., 2016 (link)). Antigen retrieval was performed with 10 mM sodium citrate buffer (pH 6.0), and samples were then treated with H2O2 (3%, 5 min), washed with 0.5% Tween 20/PBS, and blocked (5 min, RT) with Protein Block Serum-Free Solution (Agilent Dako, Santa Clara, CA). For DAB immunohistochemistry, primary antibodies (as listed in Table S1 ) were diluted in Antibody Diluent Background Reducing Solution (DAKO) and visualized with the EnVision Plus Anti-Rabbit Labeled Polymer Kit (Agilent Dako). H&E and Trichrome staining (Masson Trichrome Stain Kit from Sigma-Aldrich) were performed as previously described (Pandey et al., 2021 (link)). For fluorescent immunohistochemistry (IF-IHC), slides were incubated with indicated primary antibodies (as listed in Table S1 ) in Antibody Diluent Background Reducing Solution at 4°C overnight. After three washes with 0.05% Tween 20/PBS, Alexa Fluor 488 or 647 labeled secondary antibodies (Invitrogen) were added (1:500, RT) for 1 h with DAPI (0.5 μg/mL). LabVision PermaFluor (Thermo Fisher Scientific) was used as mounting medium. Brightfield images were captured using an Aperio ScanScope XT scanner and ImageScope software and fluorescent images were captured using a Pannoramic 250 Flash III Scanner and CaseViewer software.
Protein block serum free solution
Manufactured by Agilent Technologies
Sourced in Denmark, United States
Protein Block Serum-Free solution is a laboratory reagent designed to block non-specific binding in immunoassay applications. The solution contains a proprietary formulation that effectively reduces background signals, ensuring accurate and reliable results in various protein detection and analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Antibody diluent background reducing solution, by Agilent Technologies (8 mentions)
Target retrieval solution, by Agilent Technologies (4 mentions)
Antibody diluent, by Agilent Technologies (4 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (4 mentions)
Envision plus dual labeled polymer kit, by Agilent Technologies (3 mentions)
Vectastain elite, by Vector Laboratories (3 mentions)
Envisiontm hrp conjugated secondary antibody, by Agilent Technologies (3 mentions)
Rat anti cd86, by Abcam (2 mentions)
Alexa fluor 488 labeled anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ax 7 fluorescence microscope, by Olympus (2 mentions)
Rabbit anti iba1, by Fujifilm (2 mentions)
Immpress polymer detection kit, by Vector Laboratories (2 mentions)
Sodium citrate buffer ph 6, by Agilent Technologies (2 mentions)
Anti ps744 748 pkd antibody, by Abcam (2 mentions)
Cellsens imaging software, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Target retrieval solution ph 6, by Agilent Technologies (2 mentions)
Dakocytomation target retrieval solution ph 6, by Agilent Technologies (2 mentions)
49 protocols using protein block serum free solution
1
Immunohistochemical Analysis of Tissue Samples
2
Immunohistochemical Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Slides were deparaffinized and rehydrated as previously described30 (link). Antigen retrieval was performed in sodium citrate buffer (10 mM, pH 6.0) and tissue samples were treated with 3% H2O2 (5 min), washed with 0.5% Tween 20/PBS, and blocked with Protein Block Serum-Free Solution (Agilent, Santa Clara, CA; 5 min, RT). Primary antibodies were diluted in Antibody Diluent Background Reducing Solution (Agilent). Specific antibodies used and dilutions are listed in Supplemental Table 1 . Staining was visualized using EnVision Plus Anti-Rabbit Labelled Polymer Kit (Agilent), or biotin-streptavidin (Biocare Medical, Concord, CA) 2-step conjugation when primary goat antibodies were used. H&E staining was performed as described previously30 (link). ScanScope XT scanner and ImageScope software (Aperio, Vista, CA) were used to capture images.
3
Detecting Timp1 mRNA via RNAscope ISH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In situ hybridization (ISH) was performed using RNAscope® Assay 2.5 HD Reagent Kit-Brown (Advanced Cell Diagnostics [ACD], Hayward, CA) using the ACD mouse target probe Timp1 (NM_001044384.1, region 2-772), exactly as described in (Liou et al., 2017 (link)). The kit was used according to the manufacturer’s instructions, with an 8-min target retrieval and 15-min protease plus incubation. The mRNA signal was detected with DAB. To continue with IHC, ISH tissue samples were blocked with Protein Block Serum-Free Solution (Agilent Dako) for 30 min at RT, and then incubated overnight at 4 °C with indicated antibody diluted in Antibody Diluent Background Reducing Solution (Agilent Dako). After three washes (TBS), the slides were incubated with Rabbit AP-Polymer (Biocare Medical, Concord, CA) for 30 min at RT, rinsed with TBS (3 times), treated with Warp Red Chromogen Kit (Biocare Medical), counterstained with hematoxylin, dehydrated and mounted. Images were captured using the ScanScope XT scanner and ImageScope software.
4
Immunofluorescent Staining of NETs in Frozen Kidney Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen kidney sections were subjected to immunofluorescent staining for NETs. The sections were masked with protein block serum-free solution (Agilent, Santa Clara, CA, USA) and allowed to react with rabbit anti-citrullinated histone H3 (Cit-H3) antibody (10 μg/ml; Abcam) for 60 min at RT. The sections were made to react with Alexa Flour 594-labeled donkey anti-rabbit IgG antibody (4 μg/ml; Abcam) and FITC-conjugated mouse anti-MPO monoclonal antibody (2 μg/ml; LSBio, Seattle, WA, USA) for 60 min at RT in the dark. Autofluorescence was suppressed by Vector TrueVIEW (Vector Laboratories). After mounting with a DAPI-containing mounting solution, the sections were observed under fluorescence microscopy.
5
Immunohistochemical Evaluation of RANKL and OPG
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, paraffin sections were deparaffinized in xylene, rehydrated in graded alcohols and antigens were retrieved in citrate buffer. Endogen peroxidase was blocked using a peroxidase-blocking reagent (Dako) and non-specific binding sites were blocked by the Protein Block Serum Free solution (Dako). The slides were then incubated with the primary antibody “anti-human RANKL (70525, R&D systems) or anti-OPG (polyclonal, Abcam)”. Immunoperoxidase staining was performed using the EnVision system (Dako) according to the supplier's recommendations. Colorimetric detection was completed with diaminobenzidine (Dako) for 5 min. Slides were then counterstained with hematoxylin. RANKL and OPG expression was evaluated, as previously described, by a semi-quantitative score of the intensity and extent of the staining according to an arbitrary scale.59 (link)
6
Immunohistochemical Staining of SCD-1 and Elovl-6
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To perform immunohistochemical (IHC) staining for SCD-1 and Elovl-6, kidney sections were transferred into a Dako Target Retrieval Solution (pH 9.0; DAKO, Carpinteria, CA). The slides were microwaved at medium power for 15 min to retrieve antigens. To block endogenous peroxidase activity, the tissue sections were treated with 3% H2O2 in distilled water for 10 min, and further incubated with 5% normal goat serum at room temperature for 30 min and Protein Block Serum-Free solution (DAKO, Carpinteria, CA) for 10 min. The slides were incubated overnight with anti-SCD-1 and anti-Elovl-6 antibodies at 4 °C, and further incubated with secondary antibodies for 1 h at 37 °C. The slides were then incubated in 3,3-diaminobenzidine + H2O2 substrate and hematoxylin. The negative controls were stained under identical conditions, but with a buffer solution instead of the primary antibody. The results were viewed using an Aperio Scan Scope slide scanner.
7
Immunohistochemical Analysis of Neuronal Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed as previously described [30 (link)]. Paraffin sections were deparaffinized, dehydrated, and heated in Histofine Simple Stain MAX PO (MULTI) (Nichirei, Japan) for 20 min. After washing with distilled water, samples were placed in 1% hydrogen peroxide/methanol for 15 min to block endogenous peroxidase. Then, samples were incubated with blocking buffer (Protein Block Serum Free solution, DAKO) for 10 min at room temperature, the sections were then incubated at room temperature for 60 min in primary antibodies diluted with antibody diluent (Dako). The following primary antibodies against the antigens were used: Tuj-1 (1:300, Promega), Synaptophysin (1:400, DAKO), Nestin (1:200, Sigma-Aldrich), Ki-67 (1:100, Abcam), p53 (DAKO, 1:50). Then, they were washed three times with 0.01 M Tris buffered saline (TBS) solution (pH 7.4) and incubated with goat anti-mouse or anti-rabbit immunoglobulin labeled with dextran molecules and horseradish peroxidase (EnVision, Dako) at room temperature for 30 min. After washing with TBS, they were incubated in 3,3’-diaminobenzidin in substrate-chromogen solution (Dako) for 5-10 min. Negative controls were performed by omitting the primary antibody. The sections were counterstained with hematoxylin.
8
Immunohistochemistry of Nodose Ganglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nodose ganglia and whole brains (n = 4−5 per group) were immersed in 4% paraformaldehyde/phosphate buffer for 24 h at 4°C, incubated for 24 h in PB containing 20% sucrose, quickly frozen on dry ice and cut into 8-µm slices using a cryostat at −20°C. Sections were blocked for 5 min in protein-block serum-free solution (Dako), and then incubated overnight at 4°C with rabbit anti-Iba1 (1:10,000; Wako Pure Chemicals), rat anti-CD86 (1:100; Abcam), mouse anti-HSP72 (1:50; Enzo Life Sciences, New York, NY, USA) or mouse anti-NeuN (1:200; Millipore, Chemicon International). Immunofluorescence was performed with Alexa Fluor 488-labeled anti-rabbit secondary antibody or Alexa Fluor 594-labeled anti-mouse secondary antibody (both 1:400; Invitrogen). Images were captured on an OLYMPUS AX-7 fluorescence microscope (Olympus). Cells immunoreactive for Iba1, CD86 or HSP72 in the nodose ganglion and hypothalamus of three mice were counted manually in three to five sections per mouse using the cellSens imaging software (Olympus). Quantitation was performed in a blinded fashion.
9
Microglial Characterization in Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nodose ganglia and whole brains (n=4/group) were immersed in 4% paraformaldehyde/PB for 24 h at 4 °C, incubated for 24 h in PB containing 20% sucrose, quickly frozen on dry ice, and cut into 8-μm slices with a cryostat at −20 °C. Sections blocked for 5 min in protein-block serum-free solution (Dako, Carpinteria, CA, USA) were incubated overnight at 4 °C with rabbit anti-IBA1 (1:10 000; Wako Pure Chemicals), rat anti-CD11b (1:50; AbD Serotec, Oxford, UK), and rat anti-CD86 (1:100; Abcam, Cambridge, UK). Immunofluorescence was performed with a combination of Alexa Fluor 488-labeled anti-rabbit secondary antibody or Alexa Fluor 594-labeled anti-rat secondary antibody (both 1:400; Invitrogen). Images were captured on an OLYMPUS AX-7 fluorescence microscope (Olympus). Cells immunostained with IBA1, CD11b, or CD86 antibody were counted manually with Olympus cellSens Imaging Software (Olympus). Quantitation was performed in a blinded fashion.
10
Immunofluorescence Analysis of H. pylori and GGT in Stomach Biopsies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stomach biopsy specimens (antrum and stomach body) of H. pylori-positive patients were fixed with formalin, embedded in paraffin, and sliced into 4-μm-thick sections. Tissue sections were deparaffinized and antigen retrieval was performed via heat treatment with a Target Retrieval Solution (DAKO North America, Inc., Carponteria, CA, USA) diluted with distilled water. Thereafter, sections were incubated with Protein Block Serum Free solution (DAKO North America, Inc.) at room temperature for 20 min. An anti-H. pylori rabbit polyclonal antibody (DAKO North America, Inc.; 1:150) and a mouse monoclonal anti-human GGT antibody (Abnova, Taipei, Taiwan; 1:150) were applied as primary antibodies and incubated with the tissue sections in a humidified chamber overnight at 4 °C. The slide was washed thrice in PBS with Tween-20 (PBS-T) for 5 min each. Thereafter, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA; 1:200) and Alexa Fluor 568 goat anti-mouse IgG (Invitrogen; 1:200) were applied as secondary antibodies and incubated with the tissue sections for 1 h at room temperature in the dark. The slide was washed thrice in PBS-T for 5 min each. Fluorescence microscopic images were obtained using an All-in-One Fluorescence Microscope (BZ-X700; KEYENCE Japan, Osaka, Japan), and images were captured at × 1000 magnification.
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!