Dako buffer

The paraffin samples were cut at 2-μm thickness, deparaffinized, and rehydrated. After inactivation of endogenous peroxidase with 3% H₂O₂, slides were pre-incubated with 10% fetal calf serum in Dako buffer (Dako) to block non-specific reactions. The samples were incubated with rabbit polyclonal anti TIM-1 (10 μg/ml, ThermoFisher, PA5-20244) and goat polyclonal antibody against 8-hydroxyguanosine (8-OHdG) (2 μg/ml, Abcam, San Francisco, CA, USA, ab10802) overnight at 4°C. Anti-Nox1 (Abcam, San Francisco, CA, USA, ab131088), anti Nox2/gp91phox (Abcam, San Francisco, CA, USA, ab80508), and anti-Nox4 (Abcam, San Francisco, CA, USA, ab133302) were used to detect the various NDAPH homologues. Secondary antibodies were applied for 1 h at room temperature in 10% FCS/Dako buffer. Finally, the sections were incubated with avidin peroxidase (1:100, Sigma Aldrich) in 10% FCS/Dako buffer for 1 h at room temperature. Antibody binding was routinely visualized using DAB (Sigma Aldrich). The sections were counterstained with haematoxylin, dehydrated, and mounted in Eukitt.

For IHC, 4-mm sections were deparaffinized, rehydrated, and pretreated for 10 min in a microwave (or pressure cooker) in Dako buffer (pH 6; Dako). Samples were then incubated overnight at 4°C with the indicated primary antibodies diluted in RPMI 1640 + 10% bovine serum. Slides were washed twice with TBS/0.1% Tween 20 and then developed with the EnVision system (Dako). Immunofluorescence staining was performed in Labtek 4 chamber slides. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 2% BSA for 1 h at room temperature. Samples were incubated with primary antibody for 1 h at room temperature or overnight at 4°C. Alexa fluor secondary antibody (1:500) was used. DAPI (1 µg/mL) was used to visualize the nucleus. The Kyn antibody was tested for specificity by incubating 1:3 (10 mM Kyn + Kyn antibody) for 30 min before adding to the fixed cells.

For the analysis of TSLP expression in tissue slices, 2–4 µm thick sections were used without further pre-treatment (anti-mouse/rat TSLP; anti-Iba1), or after antigen retrieval for 1 hour in a commercial food steamer in 10 mM EDTA buffer pH 8.5 (anti-GFAP). The sections were rinsed in 0.05 M Tris-buffered saline (TBS) and incubated in Dako buffer (DakoCytomation, Vienna, Austria) containing 10% fetal calf serum (DB/FCS) for 20 min. The slides were incubated in blocking buffer at 4°C overnight with the following primary antibodies: polyclonal rabbit anti-mouse/rat TSLP (ab3, Sigma Aldrich; 1:50), polyclonal rabbit anti-Iba1 (Wako Chemicals GmbH, Neuss, Germany; 1:50), or mouse anti-rat glial fibrillary acidic protein (GFAP, Neomarkers, Fremont, CA; 1:200). The sections were then washed three times in TBS and incubated with biotinylated secondary antibodies (donkey anti-rabbit, 1:2,000, or sheep anti-mouse, 1:500; both antibodies from Jackson Immunoresearch) in DB/FCS for 1 hour at room temperature, followed by three times washing in TBS and incubation with avidin–peroxidase complex (1:100 in DB/FCS; Sigma) for 1 hour. After another washing step, labeling was visualized with 3,3′-diaminobenzidine-tetra-hydrochloride (DAB, Sigma) containing 0.01% hydrogen peroxide. All sections were counterstained with Meyer's hematoxylin, dehydrated, and mounted in Eukitt (Sigma).

Frozen bone marrow smears on plus slides were thawed on the benchtop at room temperature for 5–10 min, and then fixed in 10% NBF for 1 h. Slides were washed in TBS 2 times for 5 min, then placed in PBS before performing heat-mediated antigen retrieval in Tris-HCl buffer, pH 10, for 6 min at 75 °C. Slides were transferred to fresh PBS for 5 min to cool, and were then washed four times in Dako buffer (Dako, #K8007) by directly pipetting buffer onto each slide and decanting. Reagent 1 was then added for 30 min, followed by four washes in Dako buffer. Slides were then incubated in Reagent 2A for 40 min, followed by another set of washes, incubated in Reagent 2B for 40 min, and then washed again. Reagent 3 was spiked fresh with anti-CD11b-PE, CD14-PE, and CD34-PE at 1:200 and incubated on slides for 30 min, followed by a final set of washes. Slides were then transferred to PBS and cover-slipped with RareCyte mountant to sit overnight at room temperature in the dark.