Activation of regulated cell death in the lung of piglets infected with virulent PRRSV-1 Lena strain occurs earlier and mediated by cleaved Caspase-8

Table 2 Summary of immunohistochemical methodology

Specificity	Type of antibody	Commercial origin	Blocking solution	Dilution	Antigen retrieval
TUNEL	N.A	In Situ Cell Death Detection Kit, POD, Roche, Germany	N.A	N.A	Proteinase K in heat incubator
cCasp3 (Asp175)	mAb	SignalStain Apoptosis Cleaved Caspase-3 IHC Detection Kit, Cell Signaling, USA	5% NGS	1:500	pH 6 citrate buffer in microwave
PRRSV (SDOW 17)	mAb	Rural Technologies, Brookings, SD, USA	2% BSA	1:500	Protease XIV in water bath
CD163^* (2A10/11)	mAb	INIA, Madrid, Spain	2% BSA	Neat	pH 3.2 citrate buffer microwave
Arginase-1	pAb	ThermoFisher Scientific, Barcelona, Spain	2% BSA	1:100	pH 8.5 citrate buffer microwave
cCasp8 (Asp391)	mAb	Cell Signaling; Danvers, MA, USA	2% BSA	1:100	Proteinase K in heat incubator
cCasp9 (Asp330)	mAb	Cell Signaling; Danvers, MA, USA	2% BSA	1:50	Proteinase K in heat incubator

mAb: monoclonal antibody, pAb: polyclonal antibody, N.A.: not applicable, NGS: Normal goat serum, BSA: Bovine serum albumin (Sigma-Aldrich, Taufkirchen, Germany); Proteinase K (Roche, Basel, Switzerland), 15 min at 37 °C in heat incubator; Protease Type XIV (Sigma-Aldrich), 8 min at 37 °C in water bath. *CD163 mAb was kindly provided by Dr. J. Domínguez, INIA, Madrid, Spain.

ISSN: 1297-9716