Icc50w digital camera

Immunohistochemistry (IHC) staining was performed as previously described [8 (link)]. Briefly, tissues were fixed in 10% formalin for 36-h and embedded in paraffin or frozen-embedded in OCT solution (Tissue-Tek). Paraffin sections were prepared at a 4-μm thickness and mounted on microscope slides (Trajan Scientific and Medical, VIC, Australia). Antigen retrieval was performed at 99 °C for 20 min in 0.01 M, pH 6.0 citric buffer. Endogenous peroxidase was deactivated with 3% H2O2 (Sigma-Aldrich, Dublin, Ireland). The slides were then blocked by Protein Block Serum-Free (Dako, Glostrup, Denmark), and incubated with one of the primary antibodies, which included Fibronectin, Collagen type I (dilution 1:750, Abcam, Cambridge, UK), and 8-hydroxy-2′ -deoxyguanosine (8-OHdg, dilution 1:200, Cell Signalling Technology, MA, USA). After overnight incubation at 4 °C, biotinylated secondary anti-rabbit IgG antibodies (Dako) were incubated for 30 mins and finally horseradish peroxidase (HRP)-conjugated streptavidin (Dako) for 10 mins. Using a light microscope (Leica DM750 photomicroscope with ICC50W digital camera), six consecutive non-overlapping fields from each kidney section were photographed at 20× magnification. Image J (National Institutes of Health, USA) was used to quantitate the staining area percentage.

Amputated caudal fins of adult WT (n=3), tmem38b^-/- (n=4) and tmem38b^{Δ120-7/Δ120-7} (n=3) zebrafish were collected and fixed overnight in PFA 4% w/v in PBS. Caudal fins were stained 1 h in 0.1% w/v Sirius Red (Direct Red 80, Sigma Aldrich) in saturated aqueous solution of picric acid (Sigma Aldrich) (27 (link)). After staining, caudal fins were washed in 0.5% v/v acetic acid, and directly dehydrated three times in absolute ethanol. Samples were clarified with xylene and mounted with DPX (Sigma Aldrich). Slides were observed under polarized light with the DM2500 microscope (Leica) and acquired using the ICC50 W digital camera (Leica). Measurements were performed using Leica LAS v4.13 software on 20X images. First, the length of each actinotrichia per ray was measured by tracing a line from the most proximal red signal in the ray to the tip of the caudal fin. Then, all the actinotrichia whose length was measured were counted and the mean of the number of actinotrichia per ray was calculated.

3D7 strain of P. falciparum parasites grown in human blood (purchased from Interstate Blood Bank, USA) were used in all experiments, in agreement with approved protocols of Singapore University of Technology and Design. Synchrony of the cultures was achieved by frequent selection of ring-stage infections using sorbitol treatment. Trophozoite stage parasites (~30 hours post-invasion) were used in all experiments reported in this work.
Infected RBCs (around 5% parasitemia) were washed once in 1X PBS followed by fixation in 2% paraformaldehyde PFA for 15 min. Fixed cells were smeared on CaF₂ windows (Crystran, UK) and air-dried for further use. Samples were first examined under a 4x (NA 0.1), 10x (NA 0.25) and 40x (NA 0.65) objective lens (Leica DM750) and microscopic images were captured with a Leica ICC50W digital camera. Since the smears were not stained, the infected cells were recognized based on the typical black hemozoin dots of trophozoite stage parasites. Infected cells were selected and labeled from high contrast 40x microscopic images and the corresponding 10x and 4x images. Further scans by FTIR, O-PTIR or AFM-IR were focused on selected cells.