Anti mpo antibody

Organs from infected mice were fixated overnight at 4 °C in 4% PFA and dehydrated in an increasing ethanol row before being embedded in paraffin blocks and sectioned (4 µm). Slides were de-paraffinized using xylol and decreasing concentrations of ethanol. For Ziehl–Neelsen stainings, carbolfuchsin was used, slides were heated three times, dipped in 5% HCL in 70% Ethanol, counter-stained for 1 min with 1:10 Methylene Blue in Aqua bidest., dehydrated using increasing concentrations of ethanol and Xylol, and embedded in Entellan.
For immunohistochemical detection of MPO, antigens were retrieved by steaming in citrate buffer for 30 min. Endogenous peroxidases were blocked by a 1% H₂O₂ solution for 10 min. Blocking and permeabilization were performed by incubation with 0.1% Triton-X, 3% BSA, 5% normal goat serum for 10 min. Anti-MPO antibody (1:150, Thermo Fisher Scientific, Waltham, MA, USA) was incubated overnight at 4 °C, secondary biotinylated antibody goat α-rabbit-F(ab)2 antibody (1:500, Jackson Immuno Research, Ely UK) for 45 min at RT followed by incubations with ABC peroxidase staining kit (Thermo Fisher Scientific, Waltham, MA, USA) and DAB-staining solution (Sigma-Aldrich, St. Louis, MO, USA). Hemalaun staining was performed as described before.

The 96-well black plates were coated with anti-MPO antibody (Thermo Fisher Scientific, PA5-16672; 1:1000) overnight at 4ºC. Subsequently, the plate was washed with PBS + 0.1% Tween 20 and blocked with 2% BSA for 2 hours at room temperature. The lung tissue homogenates were obtained and centrifuged at 10,000g at 4°C for 10 minutes. The supernatant was collected and incubated overnight at 4°C. On the third day, MPO-bound DNA (NETs) was quantified using the Quant-iT PicoGreen kit (Invitrogen) as previously described (45 (link)).