Citric acid buffer

Neutrophil infiltration into tissues was assessed by immunohistochemical staining of Gr-1-positive neutrophils. Paraffin-embedded sections of lung and small intestine were dewaxed in xylene and rehydrated in a graded series of ethanol. The slides were heated at 95°C for 30 min in 0.92% citric acid buffer (Vector Laboratories, Burlingame, CA). After cooling, the slides were incubated with 2% H₂O₂/60% methanol and blocked in Tris-buffered saline containing 2.5% horse serum. The anti-Gr-1 antibody (Catalogue No: 108402; BioLegend, San Diego, CA) was applied and incubated overnight. The detection was carried out using the NovaRED substrate of an immunohistochemistry kit (Vector Laboratories). Gr-1 positive neutrophils were counted per high power field (HPF; lung ×200, intestine ×100) using ImageJ software, and the number of neutrophils per HPF was determined by averaging the counts of three HPFs.

Paraffin-embedded kidney sections were dewaxed in xylene and rehydrated in a graded series of ethanol. Slides were incubated in 0.92% citric acid buffer (Vector Laboratories, Burlingame, CA) at 95°C for 15 min. After cooling to room temperature, the slides were incubated with 2% H₂O₂ in 60% methanol and blocked in 2% normal rabbit serum/TBS, after which they were incubated with goat anti-Ki67 antibody (1:50, Santa Cruz Biotechnologies) in 1% normal rabbit serum/TBS with 0.02% Triton X-100 at 4°C overnight. The detection was carried out as per the instructions provided by an immunohistochemistry kit with NovaRED substrate (Vector Laboratories).

Liver tissues were taken from the median lobe following 24 h of reperfusion and stored in 10% formalin before being fixed in paraffin. Samples were then sectioned to 4 μm cuts and stained with hematoxylin–eosin (H&E). Liver parenchymal injury was then assessed in a blinded fashion using a semi-quantitative light microscopy evaluation. The histologic injury score for each sample was expressed as the sum of the individual scores given for five different parameters; cytoplasmic color fading, cellular vacuolization, sinusoidal congestion, necrosis, and erythrocyte stasis (15 (link)-17 (link)). Scores for each variable ranged from 0 (0%), to 1 (1%-10%), 2 (10%-30%), 3 (30%-60%), 4 (>60%), with a highest possible score of 20. Each sample score was then averaged over 10 microscopic fields. For neutrophil staining, paraffin-embedded liver tissues were dewaxed in xylene and rehydrated in a graded series of ethanol. The slides were heated in 0.92% citric acid buffer (Vector Laboratories, Burlingame, Calif) at 95°C for 30 min. After cooling, the slides were incubated with 2% H₂O₂/60% methanol and blocked in normal rabbit serum/Tris-buffered saline. The anti-Gr-1 antibody (BioLegend, San Diego, Calif) was applied and incubated overnight. The detection was carried out as per the instructions provided by an immunohistochemistry kit with NovaRED substrate from Vector Laboratories.

Renal tissue was fixed in 10% formalin and embedded in paraffin. Tissue was sectioned into 5 μm slices and stained with hematoxylin and eosin (H&E). Using light microscopy, the level of injury was assessed in a blinded fashion in the following categories: tubular cell injury, tubular cell detachment, loss of brush border, tubular simplification, and cast formation. Scores ranged from 0 (0% injury), 1 (<10%), 2 (10–25%), 3 (25–50%), 4 (50–75%), 5 (>75%), for a maximal score of 25. Scores were averaged for each sample over 10 randomly selected fields.
For Gr-1 immunohistochemistry, paraffin-embedded renal tissues were dewaxed in xylene and rehydrated in serial ethanol washes. The slides were heated in 0.92% citric acid buffer (Vector Laboratories; Burlingame, CA) at 95°C for 30 min. After cooling, the slides were incubated with 2% H₂O₂/60% methanol and blocked in normal rabbit serum/Tris-buffered saline. The anti-Gr-1 antibody (Abcam; Cambridge, MA) was applied and incubated overnight at 4°C. Vectastain ABC reagent and a DAB kit (Vector Laboratories) were used to detect the immunohistochemical reaction. Slides were counterstained with hematoxylin and examined under a phase contrast Eclipse Ti-S light microscope (Nikon; Melville, NY).

To visualize neutrophil infiltration in the liver, liver tissue sections were stained for Gr-1. Paraffin-embedded sections were dewaxed in xylene and rehydrated in a graded series of ethanol. Slides were incubated in 0.92% citric acid buffer (Vector Laboratories, Burlingame, CA) at 95°C for 15 min. Slides were then cooled to room temperature and incubated with 2% H₂O₂ in 60% methanol and blocked in 5% normal rat serum/Tris buffered saline (TBS), after which they were incubated with mouse anti-GR-1 antibody (1:50 dilution) (Cat 108402, Biolegend, San Diego CA) in 2% normal horse serum/TBS with 0.02% Triton X-100 at 4°C overnight. The detection was carried out as per the instructions provided by a commercially available immunohistochemistry kit with NovaRED substrate (Vector Laboratories). Gr-1 positive cells were counted using ImageJ software. For each section, 5 representative images were taken throughout and the number of Gr-1 positive cells was averaged across the section.

Immunohistochemistry for granulocyte-differentiation antigen-1 (Gr-1) was performed to assess neutrophil infiltration in the lungs. Paraffin-fixed lung tissues were dewaxed in xylene and rehydrated in a sequence of varying concentrations of ethanol. The slides were heated at 95°C for 30 min in 0.92% citric acid buffer (Vector Laboratories, Burlingame, CA). Slides were then cooled to room temperature before incubating in 2% H₂O₂/60% methanol and blocking in normal goat serum/Tris-buffered saline. Slides were then incubated overnight with anti-Gr-1 antibody (BioLegend, San Diego, CA), counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) and observed under a light microscope (Eclipse Ti-S; Nikon, Melville, NY). Gr-1-positive staining cells or neutrophils appearing brown were counted at 200× magnification in 3 visual fields/section. The mean was computed thereafter.

For neutrophil staining, the 10% formalin-fixed, paraffin-embedded lung tissues were dewaxed in xylene and rehydrated in a graded series of ethanol. The slides were heated in 0.92% citric acid buffer (Vector, Burlingame, CA) at 95° C for 30 minutes. After cooling to room temperature, the slides were incubated with 2% H₂O₂ in 60% methanol and blocked in 2% normal rabbit serum/Tris-buffered saline. Anti-Gr-1 antibody (BioLegend, San Diego, CA) was then applied and incubated overnight. Vectastain ABC reagent and DAB kit (Vector) were used to reveal the immunohistochemical reaction. Slides were counterstained with 4, 6-diamidino-2-phenylindole and examined under a phase contrast light microscope (Eclipse Ti-S; Nikon, Melville, NY).