In situ hybridization of 10% formalin-fixed paraffin sections was performed as described [40 (link)], using an automated Discovery System (Roche) equipped with an OmniMap anti-rabbit HRP Amplification HQ Kit, Anti-HQ Alkaline Phosphatase Multimer Kit, and ChromoMap Blue Kit (all from Roche), according to the manufacturer’s protocols. The probes for Ret, Wnt11, and Etv4 were cloned by PCR, and labeled with digoxigenin using RNA polymerase.
Automated discovery system
Manufactured by Roche
The Automated Discovery System is a laboratory instrument designed for high-throughput screening and sample analysis. It performs automated sample preparation, detection, and data processing to facilitate efficient and accurate experimental workflows.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Anti wt1, by Santa Cruz Biotechnology (1 mentions)
Anti six2, by Proteintech (1 mentions)
Alexa 488 or 568, by Thermo Fisher Scientific (1 mentions)
Bluemap kit, by Roche (1 mentions)
Anti wt1, by Abcam (1 mentions)
Paraformaldehyde solution, by Fujifilm (1 mentions)
Anti e cadherin, by BD (1 mentions)
2 protocols using automated discovery system
1
In situ Hybridization for Gene Expression
2
Immunohistochemical Profiling of Kidney Tissue
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Kidney tissues were fixed in a 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), embedded in paraffin, sectioned, and stained with haematoxylin-eosin using standard methods. Immunostaining was carried out using a BlueMap Kit and automated Discovery System (Roche) or manually for immunofluorescence staining.
The following primary antibodies were used: anti-SALL1 (Perseus Proteomics, Tokyo, Japan), anti-WT1 (Santa Cruz Biotechnology), anti-WT1 (Abcam), anti-cytokeratin (Sigma-Aldrich), anti-E-cadherin (BD Sciences), anti-SIX2 (Proteintech, Rosemont, IL), and anti-SALL1 (Abcam), which recognizes the C-terminus of SALL1. Secondary antibodies were conjugated with Alexa 488 or 568 (Thermo Fisher Scientific), and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using an LSM780 confocal microscope (Zeiss, Oberkochen, Germany).
The following primary antibodies were used: anti-SALL1 (Perseus Proteomics, Tokyo, Japan), anti-WT1 (Santa Cruz Biotechnology), anti-WT1 (Abcam), anti-cytokeratin (Sigma-Aldrich), anti-E-cadherin (BD Sciences), anti-SIX2 (Proteintech, Rosemont, IL), and anti-SALL1 (Abcam), which recognizes the C-terminus of SALL1. Secondary antibodies were conjugated with Alexa 488 or 568 (Thermo Fisher Scientific), and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using an LSM780 confocal microscope (Zeiss, Oberkochen, Germany).
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