To induce colon carcinoma, mice were injected intraperitoneally with 10 mg/kg body weight of AOM (Azoxymethane-Sigma) dissolved in saline [24 (link)]. Seven days after AOM injection, 2% DSS (Dextran sodium sulphate -Sigma) was given in drinking water for the next seven days, followed by normal water until the 21st week. At the 21st week, mice were sacrificed after 1.5 h of BrdU injection (100 mg/kg body weight). For macroscopic examinations, colons were opened longitudinally and fixed overnight in buffered 4% para-formaldehyde. For histological examination, colons were fixed overnight in buffered 4% para-formaldehyde, rolled, embedded in paraffin and sectioned (4 μm thickness). Sections were mounted on positively charged slides (Fisher Scientific) and were stained either with H&E or with anti-BrdU antibodies. Primary antibody was detected with superpicture™ kit (87-8963- Invitrogen). For apoptosis, TUNEL assay was performed as per the manufacturer’s instructions (Promega). For western blot and real time PCR, proximal colon was collected in PBS, snap frozen in liquid nitrogen and stored at −80°C.
Superpicture kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark
The SuperPicture™ Kit is a laboratory equipment product designed for imaging and analysis. It provides core imaging capabilities without interpretive commentary on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Positively charged slides, by Thermo Fisher Scientific (1 mentions)
Dextran sodium sulphate, by Merck Group (1 mentions)
Tunel assay, by Promega (1 mentions)
Fibronectin, by Abcam (1 mentions)
α sma, by Abcam (1 mentions)
Cleaved notch1, by Abcam (1 mentions)
Pparγ, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
Image pro plus image analysis software, by Media Cybernetics (1 mentions)
Microscope and camera, by Leica camera (1 mentions)
Ab18766, by Abcam (1 mentions)
Ab8934, by Abcam (1 mentions)
4 protocols using superpicture kit
1
Induction of Colon Carcinoma in Mice
2
Immunohistochemical Analysis of Kidney Tissues
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Kidney tissues were fixed in 4% PBS buffered formaldehyde, embedded in paraffin, and then sliced longitudinally into 4-μm thick sections. Antibodies against phosphorylated JNK (R & D Systems, Minneapolis, MN), cleaved Notch-1, cleaved Notch-2, α-SMA and fibronectin (Abcam, Cambridge, UK) were used for immunohistochemistry. Immunoreactivity in sections was detected using a horseradish peroxidase-3′-,3′-diaminobenzidine kit (SuperPicture™ Kit, Invitrogen, Carlsbad, CA).
3
Immunohistochemical Analysis of Renal Markers
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After being adequately perfused with PBS solutions, harvested kidneys were fixed in 4% PBS-buffered formaldehyde, embedded in paraffin, and then sliced longitudinally into 4-μm thick sections. Renal specimens were immunostained with primary antibodies against CB1R (BioWorld Tech., St. Louis Park, MN, USA), type 4 collagen, IL-1β, and PPAR-γ (Abcam, Cambridge, UK). Immunoreactivity in sections was detected using a horseradish peroxidase-3,3′-diaminobenzidine kit (SuperPicture™ Kit, Invitrogen, Carlsbad, CA, USA). Ten glomeruli in each section were randomly selected for microscopy under 200× magnification (Carl Zeiss, Gottingen, Germany). Six regions within renal glomeruli from three sections obtained from six mice were observed. The areas of positive immunolabeled regions were quantified by using Image-Pro® Plus image analysis software (Media Cybernetics, Silver Spring, MD, USA).
4
Placental PPARA and CASP12 Localization
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Peroxisome proliferator-activated receptor alpha (PPARA) and CASP12 were localised by immunohistochemistry in mouse placental tissue derived from both normal (nZ10) and obese (nZ10) fathers. In brief, paraffin sections (5 mm) were dewaxed in xylene and rehydrated through descending grades of ethanol (unless otherwise stated, all chemicals were obtained from Sigma-Aldrich). Sections underwent antigen retrieval using 0.01 mol/l sodium citrate buffer (pH 6.0) for 15 min and then incubation in the hot buffer for a further 20 min. Sections were washed for 5 min in PBS, pH 7.6. Following endogenous peroxidase quenching and blocking of non-specific binding, sections were incubated overnight at 4 8C with rabbit anti-PPARA (ab8934, Abcam, Cambridge, UK) at a concentration of 5 mg/ml in PBS or rabbit anti-caspase-12 (CASP12; ab18766, Abcam) at a concentration of 1 mg/ml in PBS. For isotype controls, primary antibody was substituted with rabbit IgG. Staining was visualised using the SuperPicture Kit (Invitrogen) followed by peroxidase substrate 3,3 0 -diaminobenzidine (DAB) (Dako, Glostrup, Denmark), and lightly counterstained with Harris hematoxylin. Sections were then dehydrated and mounted. Staining was visualised and captured using a Leica microscope and camera.
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