Blocking solution

Manufactured by Vector Laboratories

Sourced in United States

Blocking solution is a laboratory reagent used to prevent non-specific binding in immunoassays, such as Western blotting and ELISA. It works by blocking unoccupied binding sites on the solid support, reducing background signals and improving the specificity of the assay.

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Lab products found in correlation

Mounting medium, by Vector Laboratories (2 mentions) Abc reagent, by Vector Laboratories (2 mentions) Biotinylated secondary goat anti rabbit igg, by Abcam (2 mentions) Rnascope multiplex fluorescent assay, by Advanced Cell Diagnostics (2 mentions) Superfrost plus slide, by Thermo Fisher Scientific (2 mentions) 3 3 diaminobenzidine dab reagent, by Vector Laboratories (1 mentions) Centrifugal filter unit, by Merck Group (1 mentions) Elisa microplate, by Greiner (1 mentions) Biotinylated vva, by Vector Laboratories (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Mouse anti p erk, by Santa Cruz Biotechnology (1 mentions) Mab377, by Abcam (1 mentions) Mab3402, by Merck Group (1 mentions) Laminin, by Abcam (1 mentions) Fibronectin, by Abcam (1 mentions) Dab solution, by Vector Laboratories (1 mentions) Permount mounting medium, by Thermo Fisher Scientific (1 mentions) Cryostat, by Leica (1 mentions) Anti rfp, by Rockland Immunochemicals (1 mentions) Goat anti chicken 488, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Normal goat serum blocking solution (8 citations) Carbohydrate-free blocking solution (6 citations) BLOXALL Endogenous Blocking Solution (5 citations) Avidin blocking solution (4 citations) M.O.M blocking solution (3 citations) Streptavidin-biotin blocking solution (2 citations) Streptavidin blocking solution (2 citations) Blocking serum solution (2 citations) Peroxidase antigoat immunoglobulin G in blocking solution (2 citations) Endogenous peroxidase blocking solution (2 citations)

11 protocols using blocking solution

Syndecan-1 Immunohistochemical Staining

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The slides were deparaffinized and rehydrated before staining. To avoid nonspecific antibody binding, the slices were incubated in blocking solution (Vector Laboratories) for 1 h and reacted with anti-rat syndecan-1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) at a 1:100 dilution overnight at 4 °C. The slides were washed in PBS, and the sections were incubated for 1 h with biotinylated secondary goat anti-rabbit IgG (Abcam, Cambridge, UK). After incubation, ABC Reagent (Vector Laboratories) was applied to react with the biotinylated antibody for 1 h at 25 °C and attached with the 3,3′-diaminobenzidine (DAB) reagent (Vector Laboratories). Mean staining intensity without DAB for syndecan-1 was standardized as a negative control. The slides were stained with haematoxylin as a counterstain, dehydrated, and cover-slipped using mounting medium (Vector Laboratories). Images were obtained through a microscope. Syndecan-1 intensity was quantified using ImageJ software.

Seo E.H., Park H.J., Piao L.Y., Lee J.Y., Oh C.S, & Kim S.H. (2020). Immune response in fluid therapy with crystalloids of different ratios or colloid for rats in haemorrhagic shock. Scientific Reports, 10, 8067.

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VVA-Mediated ELISA for Protein Profiling

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For VVA ELISA, whole-cell lysates were prepared using RIPA-B lysis buffer. RIPA-B was exchanged with PBS using centrifugal filter units (Merck Millipore) which was repeated 4 times. 40 μg total proteins were measured using BCA protein assay kit and coated onto the ELISA microplate (Greiner bio-one, Neuburg, Germany) for 16 h at 4 °C. The total bound proteins were washed with PBS containing 0.05% Tween 20 and treated with blocking solution (Vector Labs, Burlingame, CA) for 2 h at 4 °C. Each well was washed three times and added with 10 μg ml⁻¹ biotinylated VVA (Vector Labs) for 16 h at 4 °C. Target proteins bound to biotinylated VVA were stained using Vectastain ABC kit (Vector Labs). Absorbance was measured using a microplate spectrophotometer (Berthold technologies, Wildbad, Germany) at 405 nm.

Song K.H., Park M.S., Nandu T.S., Gadad S., Kim S.C, & Kim M.Y. (2016). GALNT14 promotes lung-specific breast cancer metastasis by modulating self-renewal and interaction with the lung microenvironment. Nature Communications, 7, 13796.

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Immunohistochemical Analysis of IGF2 and Cell Markers in Rat Spinal Cord

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Ten-micrometer sections from rat L4–L6 spinal cords were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 35 min. Endogenous peroxidase activity was quenched using 3% H₂O₂ for 30 min and then non-specific binding sites were blocked by 30 min incubation in blocking solution (Vector Laboratories, Burlingame, CA, USA) at room temperature. Subsequently, the slides were incubated overnight in goat anti-IGF2 antibody (1:100; Santa Cruz Biotechnology, Dallas, TX, USA, sc-7435) at 4 °C and then for 1 h in HRP-conjugated antibody at room temperature. The IGF2 immunoreactive signal was developed using a Cy-3 Tyramide Signal Amplification (TSA) System (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. For double staining, slides were then heated in a microwave oven to remove IGF2 antibody, as described by Toth and Mezey with minor modifications [53 (link)]. Briefly, the slides were stained using mouse anti-pERK (1:100; Santa Cruz Biotechnology, sc-7383), mouse anti-GFAP (1:100; EMD Millipore, Billerica, MA, USA, MAB3402), anti-NeuN (1:25; EMD Millipore, MAB377), and mouseanti-CD11b (1:100; Abcam, Cambridge, UK, ab75476) antibodies. Finally, all signals from double staining were developed using a fluorescein isothiocyanate TSA system.

Yeh C.C., Sun H.L., Huang C.J., Wong C.S., Cherng C.H., Huh B.K., Wang J.S, & Chien C.C. (2015). Long-Term Anti-Allodynic Effect of Immediate Pulsed Radiofrequency Modulation through Down-Regulation of Insulin-Like Growth Factor 2 in a Neuropathic Pain Model. International Journal of Molecular Sciences, 16(11), 27156-27170.

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Immunohistochemical Analysis of ECM Retention

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In order to characterize the retention of native components in DEM, immunohistochemistry was used to confirm the presence of collagen type I, fibronectin, and laminin after the decellularization process. Paraffin-embedded native and DEM samples were cut into 5 µm sections and mounted onto positively charged slides. Sections were submerged in sodium citrate buffer at 95–100 °C for 20 min and placed at RT to cool for 20 min. Slides were blocked with Blocking Solution (Vectashield) and permeabilized with Tween 20-PBS. Primary antibodies collagen type I (1:500, abcam), fibronectin (1:500, abcam), and laminin (1:300, abcam) antibodies were incubated with the samples overnight at 4 °C. Samples were then rinsed and incubated in secondary anti-biotinylated binding IgG at RT for 30 min. Samples were rinsed in Tween-20/PBS and incubated in working ABC solution (Vector Lab) for 5 min at RT. Samples were rinsed and incubated with working DAB solution (Vector lab) for a few minutes until sample stained. Samples were then counterstained with hematoxylin and preserved by adding Permount mounting medium (Fisher Scientific) and a coverslip. Samples were then observed using a Nikon Eclipse TE2000 Inverted confocal microscope.

Chaitin H., Lu M.L., Wallace M.B, & Kang Y. (2021). Development of a Decellularized Porcine Esophageal Matrix for Potential Applications in Cancer Modeling. Cells, 10(5), 1055.

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Immunofluorescence and In Situ Hybridization Protocols

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For immunofluorescence analysis, mouse mandibles were dissected, fixed in 4% PFA overnight, and decalcified with 10% EDTA for 4 weeks. Then, the tissues were incubated with 15% sucrose for 2 hr and 30% sucrose overnight, followed by embedding in OCT. Frozen tissue blocks were sectioned at 10 mm on a cryostat (Leica) and mounted on SuperFrost Plus slides (Fisher). The tissue sections were blocked for 1 hr at room temperature in blocking solution (Vector Laboratories). Sections were then incubated with primary antibodies diluted in blocking solution at 4°C overnight. After washing three times with PBS, sections were incubated with secondary antibodies in blocking solution at room temperature for 1 hr. DAPI was used for nuclear staining and all images were acquired using a Keyence microscope (Carl Zeiss).
In situ hybridization was performed using RNAscope multiplex fluorescent assay (Advanced Cell Diagnostics). Briefly, tissues were fixed in 4% PFA overnight at room temperature before cryosectioning. ISH was performed on 10 μm sections according to the manufacturer’s instructions.

Jing J., Feng J., Li J., Han X., He J., Ho T.V., Du J., Zhou X., Urata M, & Chai Y. (2019). Antagonistic interaction between Ezh2 and Arid1a coordinates root patterning and development via Cdkn2a in mouse molars. eLife, 8, e46426.

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Immunofluorescence Staining of Paraffin-Embedded Tissue

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Tumor samples were fixed in 10% neutral buffered formalin, processed and
dehydrated, embedded in paraffin blocks, and mounted on glass slides. Slides
were deparaffinized, permeabilized with 0.1% triton X-100 in PBS for 20 minutes,
and treated with blocking solution (Vector Laboratories) containing 4 drops/mL
of an avidin blocking reagent (Vector Laboratories). Staining was performed with
primary antibodies including, anti-GFP (cat# ab13970, Abcam) and anti-RFP
(Rockland), which were prepared at dilutions of 1:300 each in a staining diluent
(Vector Laboratories) in conjunction with secondary antibody (1:200), goat
anti-rabbit biotin (Vector Laboratories), and detection with avidin alexa 594
(Thermo Scientific), 488-goat anti-chicken (Invitrogen), and 1:500 dilution of
DAPI. Images were captured using an Olympus BX51 microscope with an Olympus DP80
camera and CellSense Entry software.

Schweickert P.G., Yang Y., White E.E., Cresswell G.M., Elzey B.D., Ratliff T.L., Arumugam P., Antoniak S., Mackman N., Flick M.J, & Konieczny S.F. (2020). Thrombin-PAR1 signaling in pancreatic cancer promotes an immunosuppressive microenvironment. Journal of thrombosis and haemostasis : JTH, 19(1), 161-172.

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Immunohistochemical Localization of Tyrosine Hydroxylase

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Mounted sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol (100%–70%). Slides were heated to ~95 °C for 20 min in 10 mM sodium citrate buffer (pH 6.0) to unmask antigens. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide. Sections were washed in PBS and blocked with blocking solution (Vector Laboratories, Burlingame, CA). Next, sections were incubated overnight at 4 °C with rabbit anti-TH primary antibody (1:250, Millipore, Temecula, CA). Sections were rinsed in PBS and incubated with goat anti-rabbit HRP conjugated secondary antibody (1:250, Abcam, Cambridge, MA) for 1 h at room temperature. After washing in PBS, sections were incubated with DAB-peroxidase substrate solution (IHC-Tek, Ellicott City, MD) and mounted with permanent mounting medium (Vector Laboratories, Burlingame, CA). Sections were imaged on a macroscope (Wild, Heerbrugg, Switzerland) equipped with a CCD camera (Olympus, Center Valley, NJ) at either 16X (striatal sections) or 35X (SNpc sections).

Mead B.P., Kim N., Miller G.W., Hodges D., Mastorakos P., Klibanov A.L., Mandell J.W., Hirsh J., Suk J.S., Hanes J, & Price R.J. (2017). Novel Focused Ultrasound Gene Therapy Approach Noninvasively Restores Dopaminergic Neuron Function in a Rat Parkinson’s Disease Model. Nano letters, 17(6), 3533-3542.

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Spinal Cord Immunohistochemistry for MAPK Signaling

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Ten-micrometer sections from rat L4–L6 spinal cords were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 35 min, endogenous peroxidase activity was quenched using 3% H₂O₂ for 30 min, and non-specific binding sites were blocked with blocking solution (Vector Laboratories, Burlingame, CA, USA). Briefly, the slides were stained using anti-phospho-ERK1/2 conjugated Alex 488 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-JNK/SAPK conjugated Alex 488 (ThermoFisher Scientific, Waltham, MA, USA), and anti-phosphor-p38 MAPK conjugated Alex 488 (Thermo Fisher Scientific, Waltham, MA, USA) antibodies. Slides of the spinal cord were stained using anti-NeuN (EMD Millipore, Billerica, MA, USA), anti-CD11b (GeneTex, Alton Pkwy Irvine, CA, USA), and anti-GFAP (EMD Millipore, Billerica, MA, USA) conjugated rhodamine antibodies to identify neuron cells, microglial cells, and astrocytes, respectively. The cellular nuclei were identified using4′,6-diamidino-2-phenylindole (DAPI).

Lin F.Y., Huang K.F., Chen J.C., Lai M.F., Ma K.H, & Yeh C.C. (2021). The Clinical Application of Pulsed Radiofrequency Induces Inflammatory Pain via MAPKs Activation: A Novel Hint for Pulsed Radiofrequency Treatment. International Journal of Molecular Sciences, 22(21), 11865.

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Immunohistochemical Analysis of TLR4 Expression

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Toll-like receptor-4 (TLR4) is a transmembrane protein, and its activation leads to an intracellular signaling pathway and production of inflammatory cytokines, responsible for activating the innate immune system. Therefore, TLR4 was used to check the immunity in the tissues. The slides were deparaffinized and rehydrated. To avoid non-specific binding of antibody, the slices were incubated in blocking solution (Vector Laboratories, Burlingame, CA, USA) for 1 h and reacted with TLR4 rabbit polyclonal antibody (Abcam, Cambridge, UK) at a 1:100 dilution overnight at 4°C. The slides were then washed in PBS, and the sections were incubated for 1 h with biotinylated secondary goat anti-rabbit IgG (Abcam). After the incubation, the ABC Reagent (Vector Laboratories) was applied to react with the biotinylated antibody for 1 h at 25°C and attached with 3,3`-diaminobenzidine reagent (Vector Laboratories). The slides were stained with hematoxylin as a counterstain, dehydrated and cover-slipped using mounting medium (Vector Laboratories). Images were obtained under a microscope (Nikon, Tokyo, Japan). TLR4 intensity was quantified using Image J software (NIH, Bethesda, MD, USA).

Park H.J., Piao L., Seo E.H., Lee S.H, & Kim S.H. (2020). The effect of repetitive exposure to intravenous anesthetic agents on the immunity in mice. International Journal of Medical Sciences, 17(4), 428-436.

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Histological Assessment of Kidney Injury

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Kidneys from uninephrectomy and sacrifice were routinely processed, and 3µ m sections were stained for Wilms’ tumor-1 antigen (WT-1), Lotus lectin, and CD31 to assess glomerular matrix, podocyte injury, atubular glomeruli, and peritubular capillary density, respectively. Briefly, antigen retrieval was performed using microwave (750W, 5 min×3) for WT-1, Lotus lectin, and CD31, and trypsin (Sigma Aldrich, St Louis, MO, USA) for collagen IV. Endogenous peroxidase was quenched by hydrogen peroxide. Nonspecific epitopes were blocked by blocking solution (Vector Laboratories, Burlingame, CA, USA). Primary antibodies were incubated overnight at 4°C by adding rabbit anti-mouse collagen IV (1:500, EMD Millipore, Darmstadt, Germany), rabbit anti-mouse WT-1 antibody (1:200, Novus Biologicals, CO, USA), biotinylated lotus tetragonolobus lectin (1:1000, Vector Laboratories, CA, USA), and rat anti-mouse CD31 (1:50, Dianova GmbH, Hamburg, Germany). The Vectastain kit (Vector Laboratories) was used as a secondary antibody for 30 min, according to primary antibody host. Diaminobenzidine (DAB) was used as a chromogen, and sections were counterstained with hematoxylin. Slides treated with nonspecific antisera instead of primary antibody were used as negative control, and known positive tissues were used as positive controls. All slides were examined without knowing group assignment.

Lim B.J., Yang J.W., Zou J., Zhong J., Matsusaka T., Pastan I., Zhang M.Z., Harris R.C., Yang H.C, & Fogo A.B. (2017). Tubulointerstitial fibrosis can sensitize the kidney to subsequent glomerular injury. Kidney international, 92(6), 1395-1403.

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