The livers were fixed in phosphate-buffered 10% formalin for 24 h, and paraffin sections were then prepared. Immunohistochemical staining was carried out with the EnVision/HRP system (Dako, Carpinteria, CA, USA) on deparaffinized sections treated with Target Retrieval Solution (Dako). The antibodies used were as follows: anti-insulin-like growth factor 2 (IGF2) (ab9574; Abcam, Cambridge, UK), anti-α-fetoprotein (AFP) (14550-1-AP, for mouse tissues; Proteintech Group, Chicago, IL, USA), anti-AFP (A0008, for human tissues; Dako), and anti-trefoil factor 3 (TFF3) (Abbiotec, San Diego, CA, USA). 3,3′-Diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) was used for signal detection. For the detection of the TFF3 peptide, we applied signal amplification using the TSA Plus DIG Kit (PerkinElmer, Waltham, MA, USA). In situ hybridization for non-coding H19 mRNA was carried out on deparaffinized sections using the mouse H19 QuantiGene ViewRNA Probe Set (VB6-16706; Affymetrix, Santa Clara, CA, USA) and the QuantiGene ViewRNA ISH Tissue Assay Kit (Affymetrix).
Tsa plus dig kit
Manufactured by PerkinElmer
Sourced in United States
The TSA Plus DIG Kit is a laboratory equipment product designed for the detection and quantification of target analytes. It provides a reliable and efficient method for performing analyses on various samples. The core function of this kit is to facilitate the specific detection and measurement of the target analytes of interest, but a more detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine tetrahydrochloride, by Vector Laboratories (2 mentions)
Envision system hrp, by Agilent Technologies (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Ab9574, by Abcam (1 mentions)
Quantigene viewrna ish tissue assay kit, by Thermo Fisher Scientific (1 mentions)
Alexa 594 conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti ki67, by Nichirei Biosciences (1 mentions)
Anti hemagglutinin ha, by Roche (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti myc, by Abcam (1 mentions)
Cm3050, by Leica camera (1 mentions)
4 protocols using tsa plus dig kit
1
Immunohistochemical Analysis of Liver Markers
2
Dual FISH Assay for Prss56 and Tshb in Rat Brain
3
Immunohistochemical Analysis of Liver Tissue
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The livers were fixed in phosphate-buffered 4% paraformaldehyde for 24 h, dehydrated, cleared and embedded in paraffin. Immunohistochemical staining was performed using the EnVision/HRP system (DAKO, Carpinteria, CA, USA) on deparaffinized sections treated with Target Retrieval Solution (DAKO). The antibodies used were as follows: anti-GFP (A-11122, ThermoFisher Scientific), anti-p-AKT (#3787, Cell Signaling Technology, Danvers, MA, USA), anti-p-ERK1/2 (#4370, Cell Signaling Technology), anti-Myc (Abcam 32072, Cambridge, UK), anti-Ki-67 (Nichirei, Tokyo, Japan), anti-cleaved caspase-3 (#9661, Cell Signaling Technology), anti-hemagglutinin (HA) (Roche Diagnostics, Mannheim, Germany), anti-p16 (M-156) (sc-1207, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p21 (F-5) (sc-6246, Santa Cruz). The chromogen 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) and HistoGreen (LINARIS Biologische Produkte, Dossenheim, Germany) were used for signal detection. For the detection of Myc in some experiments, TSA was performed using the TSA Plus DIG Kit (PerkinElmer, Waltham, MA, USA). Morphometric analyses were performed using ImageJ software. Frozen sections were stained with Sudan III.
4
Spatial Gene Expression Analysis in Embryos
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Embryos for whole‐mount in situ hybridisation and fluorescent in situ hybridisation in sections (FISH) were fixed by 4% paraformaldehyde (PFA)/phosphate‐buffered saline (PBS) at 4°C for 12 h. Digoxigenin (DIG)‐labelled RNA probes were prepared according to the manufacturer's instructions (Roche). Whole‐mount in situ hybridisation was performed as previously described (Tonegawa et al, 1997 ). In FISH, 10‐μm‐thick frozen sections were prepared with cryostat (LEICA CM3050S). FISH protocol was described previously (Sato et al, 2008 ), with additional processes in order to amplify fluorescent signal. Additional processes are as follows. After the reaction between DIG‐labelled RNA and anti‐DIG antibody‐conjugated horseradish peroxidase (HRP; Roche), sections were washed 3 times by TNT buffer (100 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween‐20) and were treated with DIG amplification working solution in TSA plus DIG Kit (PerkinElmer) for 5 min at room temperature (RT) in accordance with manufacturer's instructions (PerkinElmer). After three times wash by TNT, samples were treated by anti‐DIG antibody‐conjugated HRP at 4°C for 12 h again. Images were processed by FIJI (https://fiji.sc ), and Fgf8 expression area on limb bud regions was measured by the “Measure” command.
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