Dab peroxidase substrate kit

Brain and muscle tissue samples of BTF-967 dog, euthanized because of a cancer development, were used to verify in vivo EGFP expression in transgenic dogs. Tissues were fixed with 4% paraformaldehyde (Sigma-Aldrich, MO, USA) and embedded in paraffin, sectioned (4μm in thickness), and placed on glass slides. After deparaffinization and hydration, tissue sections were microwaved for 20 min in sodium citrate buffer (pH 6.0) for antigen retrieval using the buffer containing 10 mM sodium citrate (SAMCHUN Chemical, Seoul, Korea) and 0.05% Tween-20 (LPS Solution, Daejeon, Korea). Endogenous peroxidase was blocked in a 9:1 mixture of methanol (DUKSAN Science, Seoul, Korea) and 30% H₂O₂ (Sigma-Aldrich) for 10 min. Then, tissue slides were stained with primary antibodies against EGFP (1:1,000; ab290, Abcam, Cambridge, UK) and RFP (1:200; 600-401-379s, Rockland Immunochemicals Inc., PA, USA), for 12 h at 4 °C. Then, tissues were washed twice with PBS and incubated with biotinylated secondary antibody (Vector Laboratories, Inc., CA, USA) for 1 h at room temperature. Finally, all samples were developed using the Vectastain ABC horse radish peroxidase (HRP) (Vector Laboratories, Inc.) and DAB peroxidase substrate kits (Vector Laboratories, Inc.) according to the manufacturer’s instructions. The tissue sections were subsequently counterstained using hematoxylin.

Mouse ovaries were fixed overnight in 10% PBS-buffered formalin, dehydrated and embedded in paraffin. Ovary samples were serially sectioned (5 μm thick) and stained with hematoxylin and eosin. GVBD rates were determined only for large preovulatory follicles that contained oocytes with clearly visible nuclei at 4 h after hCG injection.
For immunohistochemistry, sections were deparaffinized, rehydrated, and incubated with primary antibodies at room temperature for 1 h, then reacted with biotin-labelled secondary antibodies for 30 min, and finally stained using Vectastain ABC kits and DAB peroxidase substrate kits (Vector Laboratories, Burlingame, CA).

Formalin-fixed, paraffin-embedded (FFPE) tissue sections were stained for CD3 and CD8 on a Bond RX Autostainer (Leica). Antigen retrieval was performed in EDTA (pH 9.0) at 100 °C for 20 min. CD3 and CD8 antibodies (Additional file 1) were incubated at RT for 60 min (1:100,1:400) and detected with ImmPACT Red Alkaline Phosphatase and DAB Peroxidase substrate kits (Vector), respectively.

X-gal staining and whole mounting of mammary glands was carried out as previously described1 (link)7 (link)13 (link). #3 and #8 thoracic glands from the recipient nude mice were used as negative staining controls for all X-gal staining procedures. X-gal-stained whole mounts were embedded in paraffin, sectioned at 6.0 μm, and counterstained with nuclear fast red (Sigma-Aldrich, St Louis, MO, USA). Primary antibodies used were rabbit-anti-Cytokeratin 8 (ab53280 1:100; Abcam, Cambridge, MA, USA), rabbit-anti-keratin wide spectrum (Z0622 1:500; Dako, Carpinteria, CA, USA), rabbit-anti-ER alpha (sc-542 1:75; Santa Cruz Biotechnology, USA), rabbit-anti-progesterone receptor (A0098 1:150; Dako), and mouse-anti-SMA 1A4 (1:100; Life Technologies, Carlsbad, CA, USA), anti-alpha-lactalbumin31 (link) and anti-caseins32 (link). IHC staining procedure was carried out using the RTU Vectastain Universal ABC Kit and DAB peroxidase substrate kits (Vector Laboratories, Burlingame, CA, USA) per the manufacturers protocol. All sections were counter-stained with nuclear fast red or haematoxylin.

PBS-buffered formalin-fixed, paraffin-embedded samples were sectioned (5-μm-thick) for H&E staining and immunohistochemistry. To gain better histology of testis seminiferous tubules, testes were fixed in Bouin’s solution and subjected to H&E staining. Immunohistochemistry was performed as described previously^{39 (link)}. Sections were deparaffinized and rehydrated. Primary antibodies were applied at suitable dilutions (Supplementary Table 5) at room temperature for 1 h, and then incubated with biotinylated secondary antibodies for 30 min. Sections were then stained using Vectastain ABC and DAB peroxidase substrate kits (Vector Laboratories, Burlingame, CA).

Brains were removed from adult mice and post-fixed, overnight. After de-hydration, brain specimens were embedded in Paraplast Tissue Embedding Medium (Leica Biosystems, Milan, Italy) and serially sectioned to obtain sagittal sections (thickness, 9 micron) [46 (link)] that were then immunostained as previously described [38 (link)]. Briefly, sections were incubated with the anti- Aβ monoclonal antibody DE2B4 (Acris, Herford, Germany), which recognizes Aβ aggregates of senile plaques), at 4 °C overnight. After several washes with PBS, sections were incubated with biotinylated secondary antibody and antibody-antigen complexes were visualized using the Vectastain ABC Elite and DAB Peroxidase Substrate Kits (Vector laboratories, Burlingame, CA, USA), according to manufacturer’s instructions. Adjacent microscopic fields encompassing the cortical and hippocampal regions were acquired using the MetaMorph 5.5 software (Universal Imaging, Downgtown, PA, USA). The abundance of Aβ plaques per field was determined blindly and independently by two investigators and relative plaque area was measured using the MetaMorph 5.5 software tools (Universal Imaging, Downingtown, PA, USA).