Immpress hrp horse anti rabbit igg plus polymer kit

Immunohistochemical staining of CD34 and PAS and capturing of images was performed by Yale Pathology Tissue Services (YPTS). For YAP, TAZ and BIRC5 paraffin was removed from slides by three washes with xylene for 5 min, and rehydration in an ethanol gradient (100%, 100%, 95%, 80%, 70%) for 3 min each. Citrate antigen retrieval was performed in a pressure cooker for 15 min followed by cooling for 30 min under running water. Endogenous peroxidases were blocked in 0.3% hydrogen peroxide in deionized water followed by two 5-min washes with 1 × PBS. Slides were blocked with 2% horse serum in 0.1% Triton X in PBS for 1 h before an overnight incubation in a humid chamber at 4 °C with the primary antibody. The ImmPRESS HRP Horse anti-rabbit IgG PLUS Polymer Kit (Vector Labs, Cat# MP-7801) was used as per manufactures instructions. Slides were counterstained in hematoxylin and mounted with ProLong Gold (Thermo Fisher Scientific, Cat# P36934). Antibodies used are as follows: YAP (Cell Signaling Technologies, Cat.#14074; 1:1000), TAZ (Cell Signaling Technologies, Cat.#72804; 1:400), BIRC5 (Cell Signaling Technologies, Cat.#2808; 1:400) and IgG (Cell Signaling Technologies, Cat.#3900s).

Formalin-fixed paraffin-embedded (FFPE) lung tissue blocks were sectioned at 4-μm thickness by the Tissue Core at Moffitt Cancer Center. Hematoxylin and eosin (H&E)-stained sections were prepared by the Tissue Core immediately after sectioning. Immunostaining of mouse lung sections was performed overnight at 4 °C in humidified chambers with antibodies against p-Mek1/2 (Ser221) (Cell Signaling Technology Cat# 2338, RRID: AB_490903; 1:200) or p-Mapk (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology Cat# 4370, RRID: AB_2315112; 1:400) in 2.5% normal horse serum. The IHC signal was developed using DAB after conjugation with ImmPRESS HRP Horse anti-rabbit IgG PLUS polymer kit (Vector Laboratories Cat# MP-7801). Nuclei were counterstained by immersing the slides in Gill’s hematoxylin for 1 min (Vector Laboratories Cat# H-3401).

Immunostaining for pFOXO3A was carried out on ovarian cortical sections to interrogate activation of PI3K signalling in primordial and transitional follicle oocytes, according to standard protocols. Incubation with anti-pFOXO3A (ab154786, Abcam, UK) at 1:100 was carried out overnight at 4°C. ImmPRESS^® HRP Horse Anti-Rabbit IGG PLUS Polymer Kit (MP-7801, Vector Laboratories, UK) was used for secondary antibody labelling, with subsequent visualization using 3,3′-diaminobenzidine (DAB) (Agilent DAKO, USA). Tissue was incubated with DAB for 10 min, and this was kept consistent across experiments. Slides were counterstained with haematoxylin before mounting and imaged using an Axioscan slide scanner. Image analysis was carried out objectively using FIJI to measure mean grey levels of cytoplasmic pFOXO3A staining (over a scale of 0–256). Oocytes were classified as strong staining with grey levels between 0 and 120, weak staining with grey levels between 121 and 219, and no staining with grey levels between 220 and 256. These data were then processed to calculate the proportion of oocytes with strong, weak, and no pFOXO3A staining.