Ovarian sections of immunized BALB/c and unimmunized wild mice were obtained from BIOSERV Analytik (Rostock, Germany). Ovarian sections were deparaffinized and subjected to antigen retrieval using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Sections were blocked with 10% goat serum blocking solution (Life Technologies, Frederick, Maryland, USA) at RT for 1 h, washed with PBS, and incubated with 1X mouse-on-mouse IgG blocking solution (Invitrogen, Thermo Fisher Scientific) at RT for 1 h. Sections of unimmunized wild mice were incubated with a 1:20 dilution of serum samples from vaccinated BALB/c mice and kept at 4°C overnight. Sections of immunized BALB/c mice were incubated with a blocking solution (without the addition of any serum samples), washed, and incubated with 10 µg/mL fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, Thermo Fisher Scientific) at 37°C for 1.5 h. After washing, slides were mounted with the DABCO mounting medium (25 mg/mL DABCO, 90% glycerol, and 10% PBS; pH 8.5) and observed under a fluorescence microscope.
Mouse on mouse igg blocking solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Mouse-on-Mouse IgG Blocking Solution is a laboratory reagent designed to prevent cross-reactivity between mouse primary antibodies and mouse tissue or cell samples. It is used to block endogenous mouse immunoglobulins in order to improve the specificity and accuracy of immunohistochemical and immunofluorescent staining procedures.
Automatically generated - may contain errors
Lab products found in correlation
Goat serum blocking solution, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Fitc conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ab22048, by Abcam (1 mentions)
Ctxb fitc, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica camera (1 mentions)
Ab28364, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Ab22048, by Merck Group (1 mentions)
Sp8 super resolution confocal microscope, by Leica camera (1 mentions)
Ab107216, by Abcam (1 mentions)
Ma5 32 876, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
6 protocols using mouse on mouse igg blocking solution
1
Ovarian Immunohistochemistry in Mice
2
Fluorescent Immunostaining of Mouse Ovary
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Mouse ovarian sections were obtained from BIOSERV Analytik (Rostock, Germany). The sections of a wild mouse were deparaffinized and subjected to antigen retrieval using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Sections were blocked with 10% goat serum blocking solution (Life Technologies, Frederick, Maryland, USA) at RT for 1 h, washed with PBS, and incubated in 1X mouse-on-mouse IgG blocking solution (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) at RT for 1 h. Sections were incubated with a 1:20 dilution of serum samples or fecal extracts and kept at 4 °C overnight. The sections were washed and incubated with 10 µg/mL fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Invitrogen, Thermo Fisher Scientific) as the secondary antibody for 1.5 h at 37 °C. After washing, the slides were mounted with DABCO mounting medium (25 mg/mL DABCO, 90% glycerol, and 10% PBS, pH 8.5) and observed under a fluorescence microscope.
3
Lipid Raft and TLR4 Colocalization
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BV-2 cells were fixed with 4% PFA for 10 minutes and subsequently incubated with Mouse-on-Mouse IgG Blocking Solution (Invitrogen R37621) at room temperature for 30 min. Cells were incubated with recombinant Cholera toxin B (CTxB)-FITC (Sigma Aldrich C1655) for 1 h to stain for lipid rafts and then with an anti-TLR4 antibody (Abcam ab22048) at 4°C overnight, followed by an anti-mouse Alexa Fluor 594 secondary antibody for 2 h at room temperature, and slides were mounted using ProLong™ Gold Antifade Mountant with DAPI (Invitrogen P36931). Image acquisition was performed using a Leica SP8 confocal microscope and image processing using ImageJ Fiji. Colocalization assessment was executed using JACoP plugin, facilitating the calculation of Manders’ coefficients.
4
Immunofluorescence Analysis of Vascular and Smooth Muscle
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Formalin-fixed paraffin embedded tissue sections (5 µm) were blocked and stained with rabbit anti-mouse CD31 (1:200, ab28364; Abcam) and mouse anti-mouse α-SMA (1:400, 14-9760-82; Invitrogen) antibodies overnight. Mouse-on-mouse IgG Blocking Solution (Invitrogen) was used to reduce non-specific binding. The sections were washed and incubated with secondary antibodies (Alexa Fluor 555 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit, 1:200; Invitrogen) for one hour. Fluorescence was examined on a fluorescent microscope (Olympus BX51; Olympus, Tokyo, Japan). Images were taken and processed using the Infinity 3 camera (Lumenera Corporation, Ottawa, Canada) and its associated software.
5
Visualization of TLR4-Lipid Raft Assemblies
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TLR4-lipid rafts (TLR4-LR) assemblies were assessed using NaveniFlex reagents (Navinci NaveniFlex GM) according to manufacturer’s instructions. Briefly, BV-2 cells were fixed and sequentially incubated with Mouse-on-Mouse IgG Blocking Solution (Invitrogen R37621) and Navenci blocking solution at 37°C for 60 min each in a pre-heated humidified chamber. Unconjugated CTxB (for lipid raft staining, Sigma Aldrich C9903) was incubated at room temperature for 30 min and washed three times with TBS, and then stained with an anti-TLR4 (Abcam ab22048) and anti-CTxB (Sigma 227040) antibodies at 4°C overnight. The samples were incubated with a mixture of Navenibody 1 and 2 at 37°C for 60 min in a preheated humidified chamber, enzyme A in reaction buffer A for 60 min, enzyme B in reaction buffer B for 30 min, and finally, enzyme C in reaction buffer C (Texas red) for a 90 min 37°C. Cells were mounted using ProLong™ Gold Antifade Mountant with DAPI (Invitrogen P36931) and were imaged using a Leica SP8 super-resolution confocal microscope with Lightning deconvolution, and PLA-puncta were counted using the analyze particles function in Image J.
6
Immunocytochemistry for Piezo1 and β3-Tubulin
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A culture of TG and DRG neurons on glass coverslips was used for the staining. Coverslips were placed in 24 well plate. Then, cells underwent following steps: fixation in 4% PFA for 20 min, permeabilization in 0.2% Triton X-100 for 10 min, unspecific binding sites blockage in 10% normal goat serum for 30 min, blockage with Mouse-on-Mouse IgG Blocking Solution (Invitrogen, Waltham, MA, USA) for 30 min. Then, cells were incubated overnight at +4 °C with the primary antibodies, washed with PBS and incubated in secondary antibodies for 2 h at room temperature.
As primary antibodies, we used MA5-32876 (Invitrogen, Waltham, MA, USA), mouse, 1:150 dilution for Piezo1 receptors; ab107216 (Abcam, Cambridge, UK), chicken, 1:1000 dilution for β 3 tubulin, as a marker of neurons.
As secondary antibodies, we used Alexa Fluor 488 Goat anti-mouse (1:1000 dilution) to bind Piezo1 primary antibodies and Alexa Fluor 568 Goat anti-chicken (1:1000 dilution) to bind β 3 tubulin primary antibodies (both Invitrogen).
Axio Imager M2 microscope (Zeiss, Oberkochen, Germany) was used to image cells. Data were analyzed by ImageJ 1.47v (National Institute of Health, Bethesda, MD, USA).
As primary antibodies, we used MA5-32876 (Invitrogen, Waltham, MA, USA), mouse, 1:150 dilution for Piezo1 receptors; ab107216 (Abcam, Cambridge, UK), chicken, 1:1000 dilution for β 3 tubulin, as a marker of neurons.
As secondary antibodies, we used Alexa Fluor 488 Goat anti-mouse (1:1000 dilution) to bind Piezo1 primary antibodies and Alexa Fluor 568 Goat anti-chicken (1:1000 dilution) to bind β 3 tubulin primary antibodies (both Invitrogen).
Axio Imager M2 microscope (Zeiss, Oberkochen, Germany) was used to image cells. Data were analyzed by ImageJ 1.47v (National Institute of Health, Bethesda, MD, USA).
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