Dp72 microscope digital camera

Tissues were fixed in formalin, embedded in paraffin and sectioned (3 µm). Slides were deparaffinized and boiled in Citric Acid Based Antigen Unmasking Solution from Vector Laboratories (Burlingame, CA, USA) (Cat # H-3300) for antigen retrieval, blocked in 10% goat serum and incubated overnight with the following primary antibodies: Santa Cruz FL393, Cat # sc-6243, 1:300; Cell Signaling cleaved caspase-3, Cat # 9661, 1:300; Invitrogen (Carlsbad, CA, USA) CD3e, Prod # MA5-14524, 1:500; BD Biosciences (San Diego, CA, USA) CD45R, Cat # 550286, 1:500). After PBS washing, slides were incubated either with biotinylated secondary antibody and VECTASTAIN Elite ABC Reagent from Vector Laboratories (Cat # PK-7100) or with ImmPRESS HRP Reagent from Vector Laboratories (Cat # MP-7404). They were then stained with DAB substrate with hematoxylin counterstain and coverslipped. Representative images were captured at ×40. All pictures for Fig. 4c were taken at the same time with the same microscope settings. Slides were imaged using Olympus Microscope BX41 and images were acquired using Olympus DP72 Microscope Digital Camera and Olympus cellSens Imaging Software, all from Olympus Corporation (Tokyo, Japan). All photos were processed using maximize contrast, adjust intensity (for brightening), and sharpen tools. No other manipulations were done. All scale bars, 50 μm.

Tissue samples were fixed in 10% neutral buffered formalin or 4% paraformaldehyde and embedded in paraffin. Following rehydration and quenching (3% H₂O₂), antigen retrieval was performed by microwaving slides in 10 μM sodium citrate solution for 20 minutes (pH 6). Following the retrieval, slides were blocked using protein blocking solution (DAKO) and then incubated overnight at 4°C with primary antibody (Table 2). For a negative control, non‐specific rabbit IgG (DAKO) was used at the same concentration as primary antibody. Next, sections were incubated with biotinylated secondary antibodies followed by Streptavidin‐HRP. Finally, tissue sections were mounted with Surgipath Micromount^® mounting media (Leica Microsystems Inc.) and examined on an Olympus BX61 Motorized Microscope and MicroSuite^™ system (Olympus America Inc.). The operator was blinded to the treatment groups to minimize operator bias. A minimum of five fields were examined and photographed for each tissue section using Olympus DP72 Microscope Digital Camera (Olympus America Inc.). The number of positively stained immune cells were counted by the blinded operator and recorded to quantify the infiltration of immune cells into the tissue.

The SN sections from the rats were co-incubated with a monoclonal mouse anti-rat α-synuclein antibody (1 : 400, Cat. ab1903; Abcam) and a polyclonal rabbit anti-rat TH antibody (1 : 200, Cat. GTX113016; GeneTex Inc.). The secondary antibodies were a DyLight 405-conjugated donkey anti-mouse IgG antibody (1 : 100, Wuhan Antgene Biotechnology) and a TRITC-conjugated goat anti-rabbit IgG antibody (1 : 100, Kerui Biotechnology). The images were captured by an Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with an Olympus DP72 microscope digital camera. Identical illumination and camera settings were used to capture and save all the images.

Tissue preparation for fluorescent immunostaining was conducted as a standard procedure described by Rytelewska et al. [44 (link)]. In brief, prepared tissue slides of the ovaries from LW pigs were incubated were incubated overnight with rabbit polyclonal antibodies against ITLN1 (dilution 1:100), rinsed in PBS three times, and incubated with the secondary antibody (1:1000, A28175, Invitrogen, United States) for 90 min at room temperature. After that, tissue sections were rinsed in PBS three times and again dehydrated. Finally, the sections were covered with histology mounting medium Fluoroshield ™ with DAPI for nuclear counterstaining. The labelled tissue sections were analyzed with an Olympus BX51 research microscope (Olympus, Japan) equipped with an EXFO x‐Cite Series 120Q fluorescence illuminator (Excelitas Technologies Corp, United States) using appropriate filters set for DAPI and Alexa Fluor ® 488. Images were acquired with an Olympus DP72 microscope digital camera and Cell F software (Olympus, Japan). For the negative control, the primary antibody was omitted and tissues were incubated in 0.1 M PBS.

CV-IIL or RS-IIL_A22S in concentrations of 0.1 mM (calculated as tetramers) were serially diluted with the working buffer. 50 μl of 5% RBC suspension was added to 50 μl of each diluted sample and mixtures were incubated at room temperature for 1 hour. 10 μl of the last dilution, which showed complete agglutination observed by the naked eye was transferred onto the glass slide and covered by the cover glass. The cell health fitness was observed using the motorized inverted fluorescence microscope IX81 (Olympus) and images were captured using the DP72 microscope digital camera (Olympus). 10 μl of the working buffer instead of the lectin sample was used as a negative control.

Oral tissues were fixed by neutral-buffered formalin and embedded in paraffin to assess SMAD2 phosphorylation. After preparation of thin sections, slides were deparaffinised with xylene, followed by rehydration through graded alcohol to water. Antigen retrieval was performed in target retrieval solution (pH 9.0) at 120 °C for 15–20 min (S2367;  Dako). Then, endogenous peroxidase was quenched with 3% (v/v) H₂O₂ for 5 min. After blocking with 1% bovine serum albumin (BSA) at room temperature for 2 h, the sections were incubated with a primary antibody diluted with 1% BSA in phosphate-buffered saline (PBS). Slides were incubated overnight at 4 °C with primary monoclonal antibodies. Primary antibody was as follows: anti-phospho-SMAD2 (1:100, 3108; Cell signalling Technology). Slides were then incubated with horse radish peroxidase using a ChemMate EnVision kit (Dako) for 2 h and washed twice with PBS. Immunoreactivity was visualised with 0.6 nm 3,3′-diaminobenzidine tetrahydrochloride (Dojindo) and counterstained with haematoxylin. Images were acquired with a BX53 microscope and DP72 microscope digital camera (Olympus) and analysed using Olympus cellSens software. ImageJ software was used to quantify phosphorylated-SMAD2 (p-SMAD2) protein levels.

Immediately following euthanasia, entire mice were fixed in 10% neutral buffered formalin for a minimum of 48 hours. Following fixation and after evaluation via μCT, the entire hind feet of mice (ipsilateral and contralateral) were sagittally bisected and decalcified in 10% EDTA for 21–28 days. Samples were then routinely processed for histopathologic evaluation. Briefly, samples were dehydrated in increasing concentrations of ethanol and embedded in paraffin wax. Subsequently, 4–5 μm thick sections were adhered to positively charged glass slides. Sections were routinely stained with hematoxylin and eosin (H&E) for evaluation. Standard Masson’s trichrome stains were performed on select slides. All histologic evaluations were performed by a board-certified veterinary anatomic pathologist (BAG). All slides were evaluated using a Nikon Eclipse Ni-U upright microscope (Nikon Instruments Inc., Melville, New York) and digital images were obtained using an attached Olympus DP72 microscope digital camera (Olympus Corporation, Tokyo, Japan).