Flex hrp

In some PET ex vivo autoradiography experiments, the same sections (n = 10) of the pancreas as used for the autoradiography (n = 24) were stained with GLP1-R antibody (abcam, Cambridge, UK). In some other PET ex vivo autoradiography experiments, two adjacent sections (6 μm) were stained with glucagon (n = 10) and insulin (n = 10) antibodies (abcam, Cambridge, UK). Sections were fixed in pre-cooled (−20 °C) acetone for 10 min. After evaporation of acetone from the tissue sections for 20 min at room temperature, the slides were washed in phosphate buffered saline (PBS) 2 times (5 min each), incubated in 0.3% hydrogen peroxide solution at room temperature for 10 min to block endogenous peroxidase activity, washed in PBS, followed by incubation with the primary GLP-1R (ab39072), insulin (ab63820) or glucagon antibody (ab92517). The slides were washed and incubated in Flex+/HRP (Dako, Glostrup, Denmark) for 15 minutes. After another wash, the slides were developed with DAB+ (Dako, Glostrup, Denmark), counterstained with hematoxylin (Biocare Medical, San Diego, CA, USA), rinsed in water, dehydrated, and finally mounted.

Tissues were fixed in formalin and embedded in paraffin, then sections (4 µm) were cut. Following deparaffinization with xylene, the sections were rehydrated with graded ethanol and washed with PBS, then stained with hematoxylin and eosin for histological analysis. The immunohistochemistry staining of sections was performed by using the Dako EnVision FLEX kit (Dako, Glostrup, Denmark) according to the manufacturer’s instructions as previously described [44 (link)]. Briefly, the sections were subjected to antigen retrieval in Target Retrieval Solution (Dako, Glostrup, Denmark). Endogenous peroxidase was blocked with Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark). Then, the sections were incubated with primary antibody at 4 °C overnight and followed by incubation with secondary antibody FLEX/HRP (Dako, Glostrup, Denmark) at room temperatures for 30 min. Staining was developed by diaminobenzidine (DAB) substrate (Dako, Glostrup, Denmark). The stained sections were scanned by Pannoramic MIDI (3D HISTECH, Budapest, Hungary). Primary antibodies used in this study were Ki67 (Abcam, Cat# ab16667), PCNA (CST, Danvers, MA, USA, Cat# 13110), Cyclin D1 (CST, Danvers, MA, USA, Cat# 55506), E-Cadherin (CST, Danvers, MA, USA, Cat# 3195), and N-Cadherin (CST, Danvers, MA, USA, Cat# 13116).

All TMA and conventional sections were deparaffinized at 60 °C for 20 min and rehydrated in graded ethanols. Antigen retrieval was performed by immersing the tissues in citrate buffer (pH = 6.0) for 20 min in a water bath. Endogenous peroxidase and non-specific staining were blocked with 3% H₂O₂ for 20 min at room temperature. The TMA sections were incubated for 1 hour at room temperature with the following primary antibodies: PRMT1 (ab92299, Abcam, dilution: 1/200), ZEB1 (HPA027524, Sigma Aldrich, dilution: 1/500), RUNX1 (sc-365,644, Santa Cruz, dilution: 1/50), TWIST1 (ABD29 Merck Millipore, dilution: 1/100), E-cadherin (spm471, Santa Cruz, dilution: 1/100), N-cadherin (6D11, DAKO, dilution: 1/100), ß-catenin (ß-catenin-1, Dako, dilution: 1/100). Whole-mount sections were incubated with the PRMT1 (ab92299, Abcam, dilution: 1/200) and ZEB1 (HPA027524, Sigma Aldrich, dilution: 1/500). All slides were then incubated with Envision ⁱⁿ FLEX/HRP (K4063, Dako, Denmark) for 30 min. Staining patterns were visualized by exposure to 3,3′-diaminobenzidine (DAB) and counterstained with Mayer’s hematoxylin. Finally, the slides were dehydrated in ethanol, cleared in xylene and mounted for examination. Human kidney tissue was used as a positive control of staining and replacement of the primary antibody with Tris-buffered saline was used as a negative control.