Anti rabbit igg hrp

Deparaffinized and rehydrated tissue was processed for antigen retrieval using Antigen Unmasking Solution (Vectorlabs, #H3300) at 97 °C for 25 min, left to cool down for 1 h, then permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 45 min, washed with PBS and blocked with 0.2% gelatine and 0.5% bovine serum albumin (BSA) in PBS for 30 min. The tissue was incubated with polyclonal rabbit anti-mouse cleaved caspase-3 primary antibody (1:100 Cell Signalling, #9661) overnight at 4 °C, washed with PBS the next day, blocked with 0.2% gelatin and 0.5% BSA for 10 min and then incubated with HRP-anti-rabbit-IgG (Dako, # P0448) for 1 h at room temperature. Then, the tissue was washed with PBS and the 3,3’-diaminobenzidine tetrahydrochloride (DAB) solution (ThermoFischer Scientific, #34,002) was added for 10 min, following washing with tap water, haematoxylin staining and dehydration. Cleaved caspase-3 positive cells were detected using brightfield settings on a Widefield Nikon Ti2 microscope (magnification 20x) and manually quantified with Fiji by counting the brown-stained nuclei.

Nanobody recognition of recombinant VAR2CSA protein and domains was measured by ELISA. Recombinant VAR2CSA (50 nM in PBS) was coated on Nunc MaxiSorp® plates overnight at 4°C. Non-specific binding sites were blocked by incubating the plates with 5% skim milk in PBS for one hour at room temperature (RT). After three washes in PBS+0.05% Tween-20, Nbs diluted in 1% skim milk in PBS were added to the wells (50 nM) and incubated for 90 min at RT. The plates were washed three times in PBS+0.05% Tween-20 before polyclonal rabbit anti-camel Ab (kindly provided by B. Stijlemans [36] (link)) was added for 1 h (non-commercial; diluted 1∶2000 in 1% skim milk in PBS.) After three washes in PBS+0.05% Tween-20, horseradish peroxidase-conjugated (HRP) anti-rabbit-IgG (DAKO, P0448) diluted 1∶2000 in 1% skim milk in PBS was added for 1 h. The plates were washed three times in PBS+0.05% Tween-20 and binding of Nb was visualized by adding o-phenylenediamine substrate. After 20 min, the HRP enzymatic reaction was stopped by adding 2.5 M H₂SO₄ and the optical density at 490 nm measured using an ELISA plate reader (VersaMax Molecular Devices).

The protein extraction and western blot analysis protocol were described in our previous study [20 (link)]. The primary antibodies detected IGF2BP3 (#07-104) was from Merck Millipore. Other primary antibodies were from Cell Signaling (Danvers, MA) including p21 (#2946), p27 (#2552), p-Rb (Ser807/811) (#9308), cleaved-PARP (Asp214) (#9541), Cyclin D3 (#2936), CDK4 (# 12790), CDK6 (#3136) and GAPDH (#2118). The secondary antibodies were anti-Mouse IgG-HRP (Dako, 00049039, 1:30000) and anti-Rabbit IgG-HRP (Dako, 00028856, 1:10000).