Impact dab peroxidase substrate kit

Pancreatic sections (2 µm) were de-paraffinized and rehydrated in Roti-Histol (Carl Roth, Karlsruhe, Germany) and a decreasing serial solution of ethanol. Slides were heated in citrate buffer (10 mM citrate acid, 0.05% Tween 20) in a pressure cooker using a microwave for 30 min at 900 W followed by a cooling step (30 min, RT). Sections were incubated with blocking solution (5% BSA/TBST, 1 h, RT), followed by primary antibodies (1 h, RT). Rabbit anti-insulin (Abcam, Cambridge, UK, ab181547) was used as primary antibody. Hydrogen peroxide blocking was performed with a 0.3% solution (Carl Roth) for 15 min at RT, followed by incubation with HRP-labeled Goat Anti-Rabbit IgG (1 h, RT) (Abcam, ab205718). Before mounting with VectaMount Mounting Medium (H-5000), tissue sections were incubated with 3,3’-diaminobenzidine in chromogen solution (ImPACT DAB Peroxidase Substrate Kit, Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin (Carl Roth). Stained insulin in whole pancreatic sections were imaged with a MIRAX-MIDI Scanner (Carl Zeiss AG, Oberkochen, Germany), equally edited with Pannoramic Viewer and analyzed with ImageJ Software.

IHC was performed as previously described.^{28 (link)} Briefly, 10 μm paraffin sections were subjected to antigen retrieval in citrate buffer. Primary antibodies used were as follows: ERα (SP1, ThermoFisher, 1:100), PR (1294, DAKO, 1:500), CK5 (NCL-L-CK5, Leica Biosystems, 1:500), CK8/18 (NCL-L-5D3, Leica Biosystems, 1:500), and CA2 (SAB2700301, Sigma, 1:1600). Secondary antibodies were anti-Rabbit Ig MP-7401 (Vector, 1:2000; Burlingame, CA, USA) or anti-Mouse Ig MP-7402 (Vector, 1:2000). Development used the ImPACT DAB Peroxidase Substrate Kit (Vector). Images were captured using the Aperio Digital Pathology system (Leica Biosystems) and assembled in Adobe Photoshop CC with minimal linear changes to contrast. Quantification of the percentage of positive cells was performed using the Imagescope software (Leica) that used algorithms tuned for nuclear or cytoplasmic staining.

Fixed lung, liver, spleen, heart, kidney, central nervous system, and small intestine tissue specimens were routinely dehydrated and paraffin embedded. Five-micron tissue sections were stained with hematoxylin–eosin (H&E). Other sections were mounted on polarized Menzel–Gläser Superfrost plus slides (Thermo Fisher Scientific, Waltham, MA, USA) for immunohistochemistry (IHC) with a 1:200 diluted rabbit polyclonal anti-T.gondii antibody (Thermo Fisher Scientific, Fremont, CA, USA) [38 (link)]. Sections of experimentally infected mouse spleen were used as a positive control. As a negative control, the primary antibodies were omitted.
The sections were subjected to heat-induced epitope retrieval (HIER) in sodium citrate buffer (pH 6) and Peroxidase Blocking Solution (Dako REAL, Carpinteria, CA, USA). Afterwards, the primary antibody was incubated for 1 h at room temperature, followed by the addition of a secondary biotinylated goat polyvalent antibody (Thermo Fisher Scientific, Fremont, CA). Finally, DAB (ImPACT DAB Peroxidase Substrate Kit, Vector, Burlingame, CA, USA) was used as indicated by the manufacturer’s instructions. Before any steps, TBS–Tween washing solution was applied to the sections.