Dfc7000 digital camera

Prior to fixation, lungs were perfused with 10ml ice cold PBS. The heart-lung block was isolated and lungs were inflated to 20 mmH₂O with 4% PFA in PBS (Ted Pella, Redding, CA). Fixed lungs were embedded in paraffin, blocks cut into 5μm sections and stained with hematoxylin and eosin [30 (link)] (Millipore Sigma). Stained sections were imaged using a Leica DM4B fluorescent microscope with DFC7000 digital camera, and data processed in LAS X imaging software (Leica Microsystems, Wetzlar, Germany). A minimum of 20 random 400x images from each sample were used for injury scoring as described [31 (link)]. Briefly, a blinded investigator scored each image based on alveolar neutrophils, interstitial neutrophils, hyaline membrane formation, proteinaceous debris in airspace and alveolar septal thickening. To generate a lung injury score, the sum of the five variables were weighted according to relevance ascribed in [31 (link)], and normalized to number of fields evaluated. The injury score is a value between zero and one inclusive.

Human and mouse brain sections were deparaffinized, rehydrated through alcohols, and processed through antigen retrieval steps consisting of heat pretreatment in citrate buffer by either microwave or autoclave per antibody-specific protocols. Sections were treated for endogenous peroxidases with 3% hydrogen peroxide in PBS (pH 7.4), blocked in 5% non-fat milk in PBS, and incubated with primary antibodies overnight at 4 °C (see Table 2). Biotinylated secondary antibody was applied for 45 min at room temperature. Finally, sections were incubated in an avidin–biotin complex with streptavidin-HRP (Vector’s Vectastain Elite ABC-HRP kit, Burlingame, CA) and the reaction product was visualized with 0.05% diaminobenzidine (DAB)/0.01% hydrogen peroxide in PBS. Negative controls consisted of full protocol except primary antibody. The presence of neurofibrillary tangles was assessed by Gallyas silver staining using standard methods. Digital images were obtained using a Leica DM6 microscope with a DFC 7000 digital camera (Leica Microsystems, Wetzlar, Germany) and imported into Adobe Photoshop (Adobe Inc, San Jose, CA).