IHC staining for HIPPI was performed as described previously.15 (link) Formaldehyde-fixed, paraffin-embedded tissue sections were used for the IHC experiment. Tissue sections were deparaffinized, and antigen retrieval was performed in 5 mM Tris-HCl for 10 min by microwave pretreatment. Endogenous peroxidase activity was quenched with 3% H2O2, and serum was used to block non-specific binding sites with the Endogenous Biotin-Blocking Kit (Thermo Fisher Scientific, USA). Then, the slides were incubated with an anti-HIPPI monoclonal primary antibody (1:50, Mouse, Thermo Fisher Scientific, USA) at 4°C for 22 h, washed and then incubated with a biotinylated anti-rabbit lgG secondary antibody for 30 min at room temperature. Slides were visualized with diaminobenzidine and followed by counterstaining with hematoxylin. The representative photographs were taken using an Olympus BX50 microscope (Japan). Slides were evaluated using light microscopy and a standard semi-quantitative immunoreactivity score as described previously. By recording the percentage of positive staining (1<25%, 2=25%–50%, 3=50%–75%, 4≥75%) and staining intensity (0=negative, 1=weak, 2=moderate, 3=strong) for each sample, immunoreactivity score (IRS) (0–12) was calculated by multiplying positive staining percentage with staining intensity. Low and high expressions were defined according to the median IRS.
Endogenous biotin blocking kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Endogenous Biotin-Blocking Kit is designed to eliminate the interference caused by endogenous biotin in immunohistochemical (IHC) and other biotin-based assays. The kit provides a simple and effective method to block the binding of endogenous biotin, ensuring accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer a1, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm 980 confocal microscope, by Zeiss (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Bx50 microscope, by Olympus (1 mentions)
Anti cd90.2 fitc, by BioLegend (1 mentions)
Gl7 biotin, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Streptavidin cy3, by Thermo Fisher Scientific (1 mentions)
Startingblock t20 pbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Thermo Fisher Scientific (1 mentions)
Nis element, by Nikon (1 mentions)
Alexa fluor 488 tyramide superboost kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 streptavidin, by BioLegend (1 mentions)
Alexafluor 555 goat anti mouse igg h l, by Abcam (1 mentions)
Bz x710 all in one fluorescence microscope, by Keyence (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Tsa plus cyanine 3, by Akoya Biosciences (1 mentions)
18 protocols using endogenous biotin blocking kit
1
IHC Staining Protocol for HIPPI Protein
2
Quantitative RSV Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FISH-PLA was performed as previously described [33 (link)]. Briefly, at the indicated timepoints, specified cells in a 96 well glass bottom plate were fixed with 1% paraformaldehyde for 10 minutes at RT. Cells were then permeabilized with 0.2% Triton X-100 for 5 minutes at RT. Cells were blocked for endogenous biotin with an endogenous biotin blocking kit (Thermo Fisher). MTRIPs were made as described above targeting RSV genome RNA, RSV NS1 mRNA, or polyadenylated transcripts. Controls included no primary antibodies, NeutrAvidin without a V5 epitope tag, NeutrAvidin bound to a scrambled PNA oligonucleotide, NeutrAvidin bound to biotin without an oligonucleotide, no FISH, and mock infected cells. MTRIPs were then incubated with cells overnight at 37°C in a hybridization buffer of 2x SSC with 0.5% tRNA, 0.5% ssDNA and 0.2% BSA in 1x PBS. RSV genome FISH, poly(A) FISH, and associated controls were at 5 nM, while NS1 mRNA FISH and associated controls were at 10 nM. PLA was then performed as described above between the V5 tag and RSV L. Cells were positively selected for successful infection by presence of viral protein immunofluorescence. Biological replicates were used, where n = 30 cells counted.
3
Hyaluronic Acid Binding Protein Visualization
4
Fluorescence Imaging of Protein Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDCK, A549, or HeLa cells were seeded on 35 mm glass bottom dishes with 14 mm microwells (5 × 103 cells/well). After incubation at 37 °C with 5% CO2 for 24 h, the cells were fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C and subsequently permeabilized by adding 0.3% Triton X-100 for 20 min at 25 °C. The cells were blocked for the endogenous biotin using Endogenous Biotin-Blocking Kit (Thermo Fisher Scientific) according to the manufacturer's instructions and then treated with 25 μM of 15 , 16 , or 17 in 1% BSA/PBS-Tween 20 (1% BSA/PBS-T) at 4 °C for 24 h. After washing with PBS-T, primary antibodies were used to detect PHB1, PHB2, VDAC2, or ATP5B proteins, as described in Table S1 . Alexa Fluor 488–conjugated streptavidin or Alexa Fluor 546–conjugated antibodies were used as secondary antibodies. Cell nuclei were stained with diamidino-2-phenylindole (Thermo Fisher Scientific). Images were captured using a confocal laser-scanning fluorescence microscope (Nikon A1, Nikon instruments).
5
Multicolor Immunofluorescence Staining of Lymph Nodes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OCT-embedded lymph nodes were sectioned at 7 microns onto positively charged glass slides and stored at −80 °C until staining. The slides were fixed for 10 min in 75:25 acetone/ethanol at room temperature. Sections were serum blocked for 30 min at room temperature with PBS containing 10% v/v normal mouse serum, 10% v/v normal rat serum, 10% v/v normal donkey serum, 2% w/v bovine serum albumin, 0.05% w/v sodium azide, and 1 μg/ml anti-CD16/32 clone 2.4G2 (clone 2.4G2 ATCC HB-197). Biotin blocking was performed with an Endogenous Biotin Blocking Kit (ThermoFisher E21390) per the manufacturer’s instructions. Slides were then stained with a primary antibody cocktail of anti-CD90.2-FITC (1:100, clone 30-H12, Biolegend, 105306), GL7-Biotin (1:20, eBioscience, 13-5902-81), and anti-IgD-A647 (1:50, clone 11–26c.2a, Biolegend, 405708) in serum blocking buffer. Secondary stain consisted of anti-FITC-A488 (1:100, polyclonal, Life Technologies, A11090) and Strepavidin-Hilyte555 (1:20, Anaspec, 60666) in serum blocking buffer. Coverslips were mounted with Pro-Long Gold Antifade Mountant (ThermoFisher, P36930). Between each step, slides were washed with PBS 4× 30 s and dried on the grate of a BSC. Slides were cured overnight and imaged on a Zeiss Axio Observer A1 using Zen software (v2.0.0.0).
6
Immunofluorescence Staining of Membrane Receptors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured on 20×20 mm glass coverslips. At 80% confluence, they were fixed with 4% paraformaldehyde for 15 minutes and then permeabilized with 0.5% Tween 20 for 10 minutes. To reduce background signals from endogenous biotin and other non-specific binding sites, the cells were treated with Endogenous Biotin-Blocking Kit (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer's instructions, followed by StartingBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Waltham, MA) for 15 minutes. They were then incubated with 5 µg/mL of biotinylated CTB, AV or ST, together with 1:100 diluted anti-CD81 mouse monoclonal IgG1 antibodies (Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight. Cells were subsequently washed and incubated with 1:50 diluted Streptavidin-Cy3 (Thermo Fisher Scientific, Waltham, MA) with 1:500 diluted Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies (Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature. They were then washed, counterstained with Hoechst 33342, mounted and visualized using a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Inc., Oberkochen, Germany).
7
CD62L-Biotin Staining and Spatial Analysis of Memory B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenic sections were fixed with 4% PFA for 2 h with rotation at 4°C, washed four times with PBS for 10 min each, and then put in a 30% sucrose solution until sections sank to the bottom. They were then embedded in OCT compound (Fisher Scientific) and sectioned at 10 μm. An Endogenous Biotin-Blocking Kit (Thermo Fisher) followed by an Alexa Fluor 488 Tyramide SuperBoost Kit (Thermo Fisher) was used to amplify the CD62L-Biotin staining according to manufacturer’s instructions. Images were captured using a Nikon AXR confocal microscope with 40x water immersion lens. Programs used were Nikon NIS Elements and Fiji.53 (link) The Fiji Plugin that was used was Bio-Formats.54 (link) Distance from GC center was quantified by finding the central point of the GC and measuring the distance between the central point and memory B cell subsets in Fiji. GC size was quantified in Fiji.
8
Immunostaining of Epitopes and Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Epitopes were retrieved from deparaffinized sections using a heat-induced method. As described before19 , sections were alternatively bathed in boiling citrate buffer (10 mM citric acid monohydrate, pH 6.0) and Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween-20, pH 9.0). Each bathing step was repeated five times for 2 min each. After permeabilization with Triton™ X-100 (MilliporeSigma; Cat# X100) and washing with 1x PBS, sections were blocked first for endogenous biotin with Endogenous Biotin-Blocking Kit (Thermo Fisher Scientific; Cat# E21390) according to the manufacturer’s instructions and then with 2.5% goat serum in 10 mM HEPES at RT for 1 h. Sections were incubated with primary antibody anti-EPX (clone AHE-1; mouse; 1:200; Abcam; Cat# ab190715) and anti-CD68 (clone FA-11; biotin; 1:100; GeneTex; Cat# GTX43914) in 2.5% goat serum in 10 mM HEPES overnight at 4 °C. After washing in 1x PBS, sections were incubated with the secondary antibodies AlexaFluor®555 goat anti-mouse IgG H&L (1:200; Abcam; Cat# ab150114) and AlexaFluor®488 Streptavidin (1:50; BioLegend; Cat# 405235) in 10 mM HEPES at RT for 3 h. After washing with 1x PBS, sections were mounted with FluoroshieldTM with DAPI (MilliporeSigma; Cat# F6057) and covered with coverslips. Images were acquired with the All-in-One Fluorescence Microscope BZ-X710 (KEYENCE).
9
Immunohistochemical Evaluation of Splenic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens were fixed in 4% (wt/vol) paraformaldehyde (PFA) for 10 min, saturated overnight in 30% (wt/vol) sucrose at 4 °C, and embedded in Tissue-Tek optimum cutting temperature compound (Sakura) followed by freezing in −80 °C. For detection of Ly6C and BST-1, sections were fixed prior to staining with ice-cold acetone for 10 min. Sections (7 μm) were blocked with 10% (vol/vol) goat serum. Endogenous peroxidase and biotin activities were quenched respectively with 3 % (vol/vol) hydrogen peroxide solution and Endogenous Biotin-Blocking Kit (Thermo Fisher). Antibodies (listed in Supplementary Table 2 ) were diluted in PBS containing 0.05% (vol/vol) Tween-20 and 2% (vol/vol) goat serum. Primary biotinylated antibodies were visualized with HRP-conjugated streptavidin followed by TSA Plus Cyanine 3 or Cyanine 5 System (Akoya). BST-1 expressing cells were detected with anti-BST-1 antibody coupled to Alexa647 using the Lightning-Link conjugation kit (abcam). Images were acquired with ZEISS LSM 980 confocal microscope and analysed using ZEN software (both from Carl Zeiss MicroImaging).
10
Lnc-EPAV RNA FISH Assay in BMDMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For lnc-EPAV RNA FISH assay, BMDMs cultured on poly-l -lysine-coated coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, washed, and stored with 70% ethanol at −20°C. Endogenous biotin signal was blocked by using an endogenous biotin blocking kit (Thermo Fisher, USA). BMDMs were incubated with biotin-labeled lnc-EPAV probe at 50°C overnight. The cells were then incubated with Alexa Fluor 488-conjugated avidin (Thermo Fisher, USA) at room temperature for 4 h. For immunofluorescence analysis, cells were sequentially fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% bovine serum albumin (BSA), and incubated with primary antibody (mouse anti-SFPQ; Sigma, USA) followed by Alexa Fluor 546-conjugated goat anti-mouse secondary antibody (Thermo Fisher, USA). Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Images were acquired using a Carl Zeiss LSM710 microscope (objective, 40×).
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!