Paraffin-embedded rat kidney sections (4 μm thickness) were prepared by a routine procedure [17 (link)]. Sections were stained with periodic acid-Schiff reagent by standard protocol. Kidney sections were also subjected to Masson’s Trichrome staining for assessing collagen deposition and fibrotic lesions. Semi-quantitative determination of renal fibrosis score was carried out by previously reported methods [32 (link)]. Briefly, Masson trichrome-stained kidney sections were graded for the presence of interstitial fibrosis according to the following scale: 0, no evidence of interstitial fibrosis; 1, <25% involvement; 2, 25to 50% involvement, and 3, >50% involvement. The scale for each rat was reported as the mean of 10 random high-power (X400) fields per section [32 (link)]. Immunohistochemical staining was performed using established protocol, as described previously [9 (link)]. Antibodies used were as follow: rabbit polyclonal anti-α-smooth muscle actin antibody (ab5694), rabbit monoclonal anti-β-catenin antibody (ab32572), rabbit monoclonal anti-CD3 antibody (ab16669; Abcam, Cambridge, MA), mouse monoclonal anti-CD68 antibody (MCA341GA; Serotec), goat polyclonal anti-AGT (sc-7419), goat polyclonal anti-renin (sc-27320, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-AT1 receptor (AB15552; Millipore, Billerica, MA).
Ab15552
Manufactured by Merck Group
Sourced in United States, Morocco
The AB15552 is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of this product is to provide a reliable and efficient tool for various laboratory tasks. Further details on the specific features and intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab32572, by Abcam (2 mentions)
Ab75762, by Abcam (2 mentions)
Ab16669, by Abcam (1 mentions)
Mca341ga, by Bio-Rad (1 mentions)
Ab5694, by Abcam (1 mentions)
Sc 9040, by Santa Cruz Biotechnology (1 mentions)
Nb100 2289, by Novus Biologicals (1 mentions)
Quantity one analysis software, by Bio-Rad (1 mentions)
Enhanced chemiluminescence reagent, by PerkinElmer (1 mentions)
Slowfade, by Thermo Fisher Scientific (1 mentions)
Paraplast plus, by Merck Group (1 mentions)
Sc 9068, by Santa Cruz Biotechnology (1 mentions)
Ab189034, by Abcam (1 mentions)
Ab54481, by Abcam (1 mentions)
Ab15251, by Abcam (1 mentions)
Ab2893, by Abcam (1 mentions)
Vector m o m immunodetection kit, by Vector Laboratories (1 mentions)
Af1819, by R&D Systems (1 mentions)
Ab22048, by Abcam (1 mentions)
Mab1195, by R&D Systems (1 mentions)
6 protocols using ab15552
1
Histological Analysis of Rat Kidney Fibrosis
2
Quantitative Western Blot Analysis
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Western blot analysis was performed as previously described [7 (link)]. We used the following antibodies: for AHR, a rabbit anti-rat AHR antibody (1:1000, overnight incubation; NB100-2289, Novus Biologicals, Littleton, CO, USA); for angiotensin II type 1 receptor (AT1R), a rabbit anti-rat AT1R antibody (1:500, overnight incubation; AB15552, Millipore, Billerica, MA, USA); for angiotensin II type 2 receptor (AT2R), a rabbit anti-rat AT2R antibody (1:250, overnight incubation; sc-9040, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bands of interest were visualized using enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA) and quantified by densitometry (Quantity One Analysis software; Bio-Rad), as integrated optical density (IOD) after subtraction of background. The IOD was factored for Ponceau red staining to correct any variations in total protein loading. The protein abundance was represented as IOD/PonS.
3
Immunofluorescence Localization of AT1R and PLIN2 in Liver
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Fixed tissue fragments were embedded in Paraplast Plus (Sigma-Aldrich Co., St. Louis, MO, United States) and sectioned at a thickness of 5 micrometers.
Liver sections (deparaffinized and hydrated) underwent antigen retrieval using citrate buffer at pH 6.0, and were blocked in 2% glycine and 5% BSA in PBS. Sections were incubated with anti-AT1R (anti-rabbit, AB15552, 1:100, Millipore) or anti-PLIN 2 (anti-rabbit, CSB-PA920084, 1:100, Millipore), in 1% BSA in PBS for 2 h.
Subsequently, samples were treated with a secondary antibody conjugated to Alexa 488 fluorophore (anti-rabbit IgG-Alexa 488, for AT1R and PLIN 2, 1:100), and slides were mounted with SlowFade (Invitrogen, Molecular Probes, Carlsbad, CA, United States). Digital images were captured using a confocal laser scanning microscope (Nikon C2; Nikon Instruments Inc., Tokyo, Japan).
Liver sections (deparaffinized and hydrated) underwent antigen retrieval using citrate buffer at pH 6.0, and were blocked in 2% glycine and 5% BSA in PBS. Sections were incubated with anti-AT1R (anti-rabbit, AB15552, 1:100, Millipore) or anti-PLIN 2 (anti-rabbit, CSB-PA920084, 1:100, Millipore), in 1% BSA in PBS for 2 h.
Subsequently, samples were treated with a secondary antibody conjugated to Alexa 488 fluorophore (anti-rabbit IgG-Alexa 488, for AT1R and PLIN 2, 1:100), and slides were mounted with SlowFade (Invitrogen, Molecular Probes, Carlsbad, CA, United States). Digital images were captured using a confocal laser scanning microscope (Nikon C2; Nikon Instruments Inc., Tokyo, Japan).
4
Western Blot Analysis of Kidney Proteins
5
Histological Analysis of Mouse Kidney
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Paraffin‐embedded mouse kidney sections (4 µm thickness) were prepared by a routine procedure (Luo et al., 2018 ). Masson trichrome staining (BA‐4079A; BASO) and Sirius red staining (DC0040; Leagene Biotechnology) were performed according to the manufacturer's protocol. Immunohistochemical staining was performed as described previously (Zhou et al., 2017 ). After incubation with specific antibodies, kidney sections were stained using Vector M.O.M. Immunodetection Kit according to the protocol specified by the manufacturer (Vector Laboratories). For a negative control, the primary antibody was omitted, and no staining occurred. Antibodies used were as follows: Klotho (AF1819; R&D Systems), Wnt1 (ab15251; Abcam), AT1 (AB15552; Merck Millipore), ACE (ab75762; Abcam), AGT (SAB2100072; Sigma‐Aldrich), p16INK4A (ab189034; Abcam), γ‐H2AX (ab2893; Abcam), PGC‐1α (ab54481; Abcam), and DKK1 (BM4554; Boster).
6
Retinal Immunohistochemistry Protocol
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Retinas were examined by immunostaining as previously reported.17 (link) Immunohistochemistry was performed using the following primary antibodies: AT1-R (1 : 100; AB15552, Merck Millipore, Billerica, MA, USA), TLR4 (1 : 100; ab22048, Abcam, Cambridge, MA, USA), GS (1 : 500; MAB302, Merck Millipore), GLAST (1 : 1000; Rb-Af660, Frontier Institute, Hokkaido, Japan), βIII tubulin (1 : 100; MAB1195, R&D, Minneapolis, MN, USA and 1 : 100; 5568, Cell Signaling, Danvers, MA, USA) and iNOS (1 : 100; 610328, BD Biosciences, San Jose, CA, USA).
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