Free-floating fixed sections were blocked in 5% goat serum, 0.5% Triton X-100 (TX) in PBS for 1 h at room temperature, and then were incubated with primary antibody in 1.25% goat serum and 0.125% TX in PBS for 48 h at 4°C. Sections were then washed 3× with PBS and incubated with secondary antibodies in 1% goat serum and 0.1% TX in PBS for 2 h at room temperature. Finally, sections were washed 3× with PBS and mounted in Vectashield Antifade (Vector Laboratories) or Prolong Diamond (Thermo Fisher Scientific) mounting medium. Sections stained with mouse primary antibodies were blocked in ReadyProbes “Mouse on Mouse” IgG blocking solution (Thermo Fisher Scientific) for 1 h before the initial blocking step. Primary antibodies and dilutions used were rabbit anti-calretinin (CR; 1:2000; Swant), mouse anti-calbindin (CB; 1:500; Swant), and rabbit anti-neuropeptide Y (NPY; 1:2000; Immunostar). Secondary antibodies used were Alexa Fluor 647 anti-rabbit (1:1000; Thermo Fisher Scientific) and Alexa Fluor 647 anti-mouse (1:500; Thermo Fisher Scientific).
Readyprobes mouse on mouse igg blocking solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
ReadyProbes "Mouse on Mouse" IgG blocking solution is a laboratory reagent designed to prevent non-specific binding of mouse immunoglobulin (IgG) antibodies in immunohistochemistry and immunofluorescence applications involving mouse-derived primary antibodies on mouse tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield antifade, by Vector Laboratories (2 mentions)
Anti rabbit alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Anti mouse alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Prolong diamond, by Thermo Fisher Scientific (2 mentions)
Rabbit anti calretinin cr, by Swant (1 mentions)
Mouse anti calbindin cb, by Swant (1 mentions)
Rabbit anti calretinin, by Swant (1 mentions)
Mouse anti calbindin, by Swant (1 mentions)
Rabbit anti neuropeptide y, by Immunostar (1 mentions)
Can get signal immunostain solution b, by Toyobo (1 mentions)
Liberate antibody binding solution, by Polysciences (1 mentions)
Bz x810 microscopy, by Keyence (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Ma1 16504, by Thermo Fisher Scientific (1 mentions)
A32794, by Thermo Fisher Scientific (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
4 protocols using readyprobes mouse on mouse igg blocking solution
1
Immunofluorescence Labeling of Calretinin, Calbindin, and NPY
2
Immunofluorescent Staining of Neuronal Markers
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Free-floating fixed sections were blocked in 5% goat serum, 0.5% Triton X-100 (TX) in PBS for 1 hour at room temperature, and then incubated with primary antibody in 1.25% goat serum, 0.125% TX in PBS for 48 hours at 4°C. Sections were then washed 3x with PBS and incubated with secondary antibodies in 1% goat serum, 0.1% TX in PBS for 2 hours at room temperature. Finally, sections were washed 3x with PBS and mounted in Vectashield Antifade (Vector Laboratories) or Prolong Diamond (Molecular Probes) mounting medium. Sections stained with mouse primary antibodies were blocked in ReadyProbes “Mouse on Mouse” IgG blocking solution (Thermofisher) for 1 hour before the initial blocking step. Primary antibodies and dilutions used were rabbit anti-calretinin (Swant, 1:2000), mouse anti-calbindin (Swant, 1:500), and rabbit anti-neuropeptide Y (Immunostar, 1:2000). Secondary antibodies used were AlexaFluor 647 anti-rabbit (Thermofisher, 1:1000) and AlexaFluor 647 anti-mouse (Thermofisher, 1:500).
3
Histological Analysis of Mouse Tissue
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Tissue samples from the mice were fixed with 4% paraformaldehyde for 24 h, decalcified in 12% EDTA for 10 d, embedded in paraffin, sectioned, deparaffinized and stained with hematoxylin and eosin (H&E) and Alcian blue. For IHC staining, antigen retrieval was performed via incubation with Liberate Antibody Binding Solution (Polysciences, USA) for 20 min at RT. Non-specific antigens were blocked using Blocking One (Nacalai Tesque) for 1 h at RT. For antibodies generated from the mouse host, tissues were blocked with ReadyProbes™ Mouse on Mouse IgG Blocking Solution (Invitrogen) for 60 min at RT. Antibodies (diluted in Can Get Signal Immunostain Solution B, TOYOBO) were incubated overnight at 4℃. After rinsing in PBST buffer, samples were incubated with Alexa Fluor secondary antibodies diluted in Can Get Signal Immunostain Solution B for 1 h at RT. A mounting medium with DAPI (Vector Laboratories) was used to counterstain the nuclei. Images were captured using a BZ-X810 microscopy (Keyence, Japan). The antibodies for IHC are listed in Additional file 1 : Table S3.
4
Dual IF Staining of HIF-1α and Vimentin
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Sections of harvested tumor tissue were used for the assessment of HIF-1a and vimentin expression with double immunofluorescence (IF) staining. The protocol of the staining and analysis was analogous to the one used with vascular morphology and perfusion assessment, but an additional step of 60 min of blocking with ReadyProbes™ Mouse-on-Mouse IgG Blocking Solution (R37621, Invitrogen, USA) was added before incubation with primary antibodies. Immunohistochemical staining was performed with mouse anti-HIF1a (MA1-16504, Invitrogen, 1:200) and rabbit anti-vimentin (SAB5700070, Sigma-Aldrich, 1:200) antibodies applied for 90 min at 37 °C. Immunodetection was performed with donkey polyclonal anti-rabbit combined with Alexa Fluor 555 and goat polyclonal anti-mouse combined with Alexa Fluor 488 (A32794, A11001, 1:200, Invitrogen, USA) antibodies incubated for 45 min in room temperature. Photos of the slides were taken with the Nikon Eclipse Ti and dedicated software (version 7.8.3). The photos were analyzed using a script written in the Matlab environment. After enhancing contrast, binarization, and background subtraction, the intensity and density of the stained area in each channel were calculated.
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