Pyelonephritis in slaughter pigs and sows: Morphological characterization and aspects of pathogenesis and aetiology

Isling, Louise K; Aalbæk, Bent; Schrøder, Malene; Leifsson, Páll S

doi:10.1186/1751-0147-52-48

Acta Veterinaria Scandinavica

Table 1 Immunohistochemical staining procedures.

From: Pyelonephritis in slaughter pigs and sows: Morphological characterization and aspects of pathogenesis and aetiology

Antibody specificity	Clone	Source/cat. no.	Dilution¹	Washing²	Blocking of endogenous peroxidase activity³	Blocking of unspecific protein binding⁴	Antigen retrieval⁵	Detection⁶	Chromogen⁷/min	Nonsense antibody⁸
Monoclonal
Mouse anti-porcine CD3ε	PPT3	SouthernBiotech, Inc USA/ SB 4510-01	1:1000^a	TBS +0.5% Triton-X-100	0.6% H₂O₂	Ultra V Block^a	Tris-EGTA^a	Ultra vision ONE HRP Polymer^a	DAB^a/6	X0931
Mouse anti-human CD79αcy	HM57	Dakocytomation, Denmark/ M7051	1:50^c	TBS +0.5% Triton-X-100	0.6% H₂O₂	Ultra V Block^a	Tris-EDTA^b	Ultra vision ONE HRP Polymer^a	DAB^a/10	X0931
Mouse anti-human monocyte, macrophage, neutrophil	MAC387	Serotec Ltd, UK/MCA874G/ MAC387	1:500^d	TBS	0.6% H₂O₂	Ultra V Block^a	Tris-EDTA^b	UltraVision LP large volume detection system HRP polymer^b	AEC^b/10	X0931
Polyclonal
Goat anti-pig IgG-Fc fragment		Bethyl Laboratories, USA/ A100-104A	1:7000^c	TBS	0.6% H₂O₂	5% rabbit serum^b	Protease^c	PAP-goat^c	DAB^a/6	I9140
Goat anti-pig IgA		Bethyl Laboratories, USA/ A100-102A	1:4000^c	TBS	0.6% H₂O₂	5% rabbit serum^b	Protease^c	PAP-goat^c	DAB^a/6	I9140
Goat anti-pig IgM μ- chain specific		Bethyl Laboratories, USA/ A100-100A	1:5000^c	TBS	0.6% H₂O₂	5% rabbit serum^b	Protease^c	PAP-goat^c	DAB^a/6	I9140
Rabbit anti-human Lysozym		Dakocytomation, Denmark/ A0099	1:200^a	TBS +0.5% Triton-X-100	3% H₂O₂	Ultra V Block^a	0.1% trypsin^e	Ultra vision ONE HRP Polymer^a	DAB^a/10	X0903
Rabbit anti- Escherichia Coli		Dakocytomation, Denmark/ B0357	1:500^e	TBS	0.6% H₂O₂	Ultra V Block^a	Protease^d	Ultra vision ONE HRP Polymer^a	AEC^b/10	X0903
Sheep anti-human Uromucoid (IgG fraction)		The Binding Site Ltd, UK/ PC071	1:700^b	TBS +0.5% Triton-X-100	0.6% H₂O₂	5% rabbit serum^b	Protease^c	AP-sheep/goat^d	DAB^a/6	013-000-002

^{1 ab} Diluted in ^a0.1% or ^b1% bovine serum albumin (BSA) (A7906, Sigma-Aldrich A/S Denmark) and 0.01% Tween 20 (P-1379, Sigma-Aldrich A/S, Denmark) in Tris-buffered saline 0.05 M Tris, pH 7.6, 0.15 M NaCl (TBS). ^cdDiluted in ^c0.1% or ^d2% BSA in TBS ^eDiluted in 5% normal swine serum (26250-084, Invitrogen, Denmark) in TBS.
²Triton X-100 (PIER28314, VWR - Bie & Berntsen A/S, Denmark).
³15 min at room temperatur.
^{4 a}Ultra V Block from Ultra vision ONE HRP Polymer (TA-125-UB, Thermo Scientific, USA). ^bRabbit serum (X0902, Dakocytomation, Denmark).
^{5 ab} Microwave oven in ^aTris-EGTA/^bTris-EDTA buffer pH 9 2 × 5 min (watt 700) and 15 min cooling. ^cd Protease (P8038, Sigma-Aldrich A/S, Denmark) (^d0.18-^c0.36 mg/ml) in TBS followed by incubation in ice cold TBS (pH 7.6) for 2 × min. ^eTrypsin 1 mg/ml (Sigma-Aldrich A/S, Denmark) in TBS for 2 h at 37°C.
^{6 a} UltraVision ONE large volume detection system HRP polymer (Ready-to-use) (TL-125-HLJ,
Thermo Scientific, USA). ^bUltraVision LP large volume detection system HRP polymer (Ready-to-use) (TL-125-HL, Thermo Scientific, USA). Both systems were applied according to the manufacturers' instructions. ^cA three layer horseradish PAP (peroxidase anti-peroxidase) technique using Z0454 (Dakocytomation, Denmark) (1:200 in TBS, 30 min RT) and P1901 (Sigma-Aldrich A/s, Denmark) (1:100 in TBS, 30 min RT). ^dDonkey Anti-Sheep/Goat immunoglobulins (peroxidase conjugate) (AP360, The Binding Site Ltd, UK).
^{7 a}DAB (4150, Kem-En-Tec A/S, Denmark). ^bAEC-Ready solution from PowerVision+ Poly-AP IHC Kit (Immunovision Techonologies, co). Counterstained with Mayers Haematoxylin 10 sec (LAB00254, VWR - Bie & Berntsen A/S, Denmark).
⁸Togheter with primary antibodies parallel sections were run with nonsense matching species polyclonal (X0903, Dakocytomation, Denmark; I9140, Sigma-Aldrich A/S, Denmark; 013-000-002, Jackson ImmunoResearch, UK) or monoclonal isotype (X0931, X0943, X0944 Dakocytomation, Denmark) antibody in the same protein concentration as the primary antibody.

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ISSN: 1751-0147

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