Organs (pancreas, kidney and liver) from all experimental groups were fixed in buffered formalin (10%) for 24 h; followed by dehydration in decreasing concentration of alcohol, infiltration, embedding and staining with hematoxylin and eosin stain51 (link). Immunohistochemical studies were performed according to Farid et al.29 (link). Where, four μm long pancreatic sections, formalin fixed, were deparaffinized, rehydrated, and washed by saline. The activity of endogenous peroxidases was blocked by adding 3 percent H2O2/methanol to pancreatic tissues, followed by washing with saline and blocking with bovine serum albumin (5%). Anti-insulin antibody (Sigma I2018) and anti-NF-kB p65 antibody (Abcame ab86299) were applied as primary antibodies for NF-kB and insulin, respectively. After 60 min of incubation, pancreatic sections were washed and treated for 120 min with HRP-conjugated rabbit anti-rat IgG (Abcam ab6734) as a secondary antibody. To visualize the response, 3,3-diaminobenzidine (DAB) was employed till a brown colour developed. Pancreatic sections were counterstained using Mayer's hematoxyline.
Hrp conjugated rabbit anti rat igg
Manufactured by Abcam
HRP-conjugated rabbit anti-rat IgG is a secondary antibody that recognizes rat immunoglobulin G (IgG). The antibody is conjugated with horseradish peroxidase (HRP), an enzyme that can be used for signal detection in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Histovt one, by Nacalai Tesque (1 mentions)
Blocking one histo, by Nacalai Tesque (1 mentions)
Fitc conjugated goat anti rat igg, by Proteintech (1 mentions)
Vector trueview autofluorescence quenching kit, by Vector Laboratories (1 mentions)
Hrp conjugated rabbit anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated swine anti rabbit igg, by Agilent Technologies (1 mentions)
3 protocols using hrp conjugated rabbit anti rat igg
1
Histological and Immunohistochemical Analysis of Organs
2
Immunohistochemical Analysis of p-AMPKα and Mac-2
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Sections were immersed in HistoVT One (Nacalai) for antigen activation. Sections were permeabilized with 0.1% Triton X-100 in tris buffered saline for staining of intracellular proteins. Sections were blocked with 0.3% H 2 O 2 in methanol and Blocking One Histo (Nacalai). Sections were incubated with anti-p-AMPKα (T172) rabbit antibody (Cell Signaling Technology) and anti-Mac-2 rat antibody (Biolegend) at 4°C overnight. The secondary antibody was HRP-conjugated goat anti-rabbit IgG (Cappel) and HRP-conjugated rabbit antirat IgG (Abcam). The Peroxidase Stain diaminobenzidine kit (Nacalai) was used as a chromogenic agent.
For immunofluorescence staining of Gr-1-positive cells, sections were performed antigen activation, subsequently blocked with Blocking One Histo (Nacalai). Sections were incubated with anti-Gr-1 rat IgG (Biolegend) at 4°C overnight. The secondary antibody was FITC-conjugated goat anti-rat IgG (Proteintech). Sections were incubated with Vector TrueVIEW Autofluorescence Quenching Kit (Vector) for reducing the autofluorescence emission.
For immunofluorescence staining of Gr-1-positive cells, sections were performed antigen activation, subsequently blocked with Blocking One Histo (Nacalai). Sections were incubated with anti-Gr-1 rat IgG (Biolegend) at 4°C overnight. The secondary antibody was FITC-conjugated goat anti-rat IgG (Proteintech). Sections were incubated with Vector TrueVIEW Autofluorescence Quenching Kit (Vector) for reducing the autofluorescence emission.
3
Immunostaining Techniques for Osteopontin, FGF23, FGFR1, and Klotho
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After the treatment with 0.3 % hydrogen peroxidase and 1% BSA-PBS, the sections were incubated with rabbit anti-osteopontin antisera (No. LB-4225, Cosmo Bio, Co., Ltd., Tokyo, Japan) at a dilution of 1: 2000 for 1 h, followed by incubation with HRPconjugated swine anti-rabbit IgG (DakoCytomation). The immunoreacted sections were subjected to the detection of TRAP enzymatic activity as previously described [22] . They were then incubated with rat anti-FGF23 (R&D systems) at a dilution of 1: 100 for 2 h and then with HRP-conjugated rabbit anti-rat IgG at a dilution of 1: 100 for 1 h. For the detection of FGFR1 and klotho, the specimens were incubated with mouse anti-FGFR1 (Abcam PLC) at a dilution of 1: 25 or mouse anti-mouse klotho antibody (Thermo Fisher Scientific Ltd.) at a dilution of 1: 50 for 2 h. These sections were then reacted with HRP-conjugated rabbit anti-mouse IgG (Invitrogen Co. A) at a dilution of 1: 100 for 1 h. For visualizing the immunoreaction, DAB was used as the substrate, and the specimens were faintly counterstained with methyl green before observation under a light microscope.
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