Lsm 710 laser

Positive transgenic P. tricornutum colonies were inoculated into f/2 liquid medium containing 100 μg/mL Zeocin for one week of growth. The green fluorescence signals were observed and recorded under a ZEISS LSM710 laser scanning confocal microscope (Carl Zeiss, Jena, Germany) with an excitation wavelength of 488 nm and emission wavelength of 510–540 nm for eGFP and 625–720 nm for chloroplast autofluorescence.

Cardiomyocytes were visualized by iFluor 594-labeled phalloidin (1:1000, Abcam, UK) staining actin and ECM was visualized by staining collagen III (ab 7778 1:400, Abcam, UK). Each slide was objected to 10 images. Morphometric analysis was performed using the software Fiji (http://fiji.sc). An average area value from each heart was calculated by the use of the measurements of 30–50 cells containing a central nucleus. Statistical analysis was performed using a two-tailed Student t test assuming unequal variances. Each pixel was calibrated to 0.83 μm according to Zeiss confocal data. Fluorescence images were collected at 20× objective using Zeiss LSM 710 laser-scanning confocal microscope (Zeiss, Oberkochen, Germany).

Tissues were fresh frozen in OCT Cryomount (Histolab, 45830) and 5 µm sections were stained following the RNAscope Fluorescent Multiplex Assay (Advanced Cell Diagnostics, USA) instructions. Images were acquired with a Zeiss LSM 710 laser scanning microscope.

Image acquisition and neuronal activation analysis was performed on a Zeiss LSM710 laser scanning confocal microscope. Double-labeled images from the regions of interest—OFC, PrL, NA core and NA shell—were obtained using appropriate filter sets. Analysis of neuronal activation was performed by counting cFos-positive nuclei, in corresponding areas in two sections/region of interest/mouse (bregma 2.22/2.68 for OFC, bregma 1.94/2.34 for PrL, bregma 1.34/1.78 for NA core, and bregma 1.10/1.42 for NA shell; Franklin and Paxinos, 2008 ), by two independent observers unaware of genotype; Pearson’s r correlation (inter-reliability) between observers was r = 0.32, p < 0.01. Neuronal activation in each region was averaged over observers and subjected to statistical analyses.

The calvariae were decalcified in 10% EDTA solution, embedded in paraffin, and sectioned into 5- to 10-μm sections. Hematoxylin and eosin (H&E) staining was performed as per previously published protocols (Huang et al., 2020 (link)).
For immunofluorescent staining, the slides were pre-treated with 5% BSA blocking buffer for 1h at room temperature and stained for osteomarkers and macrophage-specific antigens using mouse monoclonal [65529.111] anti-bone morphogenetic protein 2 (BMP2) antibody (1/100, ab6285, Abcam), mouse monoclonal [OCG3] anti-osteocalcin (OCN) antibody(1/100, ab13420, Abcam), rabbit monoclonal anti-iNOS antibody (1/100, ab15323, Abcam), rabbit monoclonal anti-CD206 antibody (1/100, ab64693, Abcam), and mouse monoclonal [3A6] anti-IL-1β antibody (1/100, 12242, Cell Signaling). Sections were then stained with anti-mouse FITC and anti-rabbit TRITC secondary antibodies (1/200, Sigma), imaged using Zeiss LSM 710 laser scanning confocal microscope equipped with Zen image analysis software. For a vascular marker, sections were stained with rabbit polyclonal anti-CD31 antibody (1/100, ab28364, Abcam) and peroxidase-conjugated secondary antibody. ImageJ was used to measure positive immunostained cell number or % area per field (n = 4 per group). The positive cell number of iNOS and CD206 was divided by the number of nuclei per field.

HCT116 cells were fixed with methanol for 2 h and blocked with 5% BSA for 60 min at room temperature. After blocking, cells were incubated with FITC-P-LPK or anti-SLC1A5 (Abcam, Cat No. ab237704, 1:50) at 4 °C overnight. Then, anti-SLC1A5 cells were further incubated with APC conjugated anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, Cat No. A-10931, 1:250) for 20 min at room temperature in a lucifugal chamber. Samples were washed with PBS for three times followed by treatment with DAPI. Samples were photographed using Zeiss LSM 710 laser confocal scanning microscopy.