Axiocam monochrome camera

Epifluorescence images of the in vitro hematopoietic cultures and immunostainings were taken using the 10x and 20x objectives of a Zeiss AxioObserver Z1 microscope and a Zeiss AxioCam monochrome camera and were processed with the Zen Blue software. Confocal images of the in vitro hematopoietic cultures were taken with a Zeiss LSM780 microscope using a 10x objective and processed with Zen Black software. All images were exported as separate layers in JPEG format and assembled in Adobe Photoshop when required. Brightness and contrast adjustments were applied equally to all images.

C3H/HeJ mice were infected with strains of C. rodentium as described above. Mice were euthanized on day 9 post infection, and the third most distal centimeter of the colon was fixed in 10% neutral buffered formalin. The tissue was paraffin-embedded and cut into 4 μm sections. The slides were deparafinized in xylene twice for 5 min, followed by rehydration in a gradient of 100% ethanol twice for 5 min, 95% ethanol for 5 min, 70% ethanol for 5 min, and dH₂O for 5 min. The samples were then boiled in 1.8 mM citric acid and 8.2 mM sodium citrate in dH₂O for 10 min, for antigen retrieval. The slides were left to cool down at RT for 10 min in the buffer, and were subsequently washed with PBS containing 0.2% Tween 20. Further, the samples were blocked in PBS containing 0.2% Tween 20, 10% FBS, and 3% BSA for 1 h at 37°C. The samples were stained with anti-Citrobacter LPS rabbit polyclonal antibody in PBS containing 0.2% Tween 20 and 3% BSA at 4°C for 3 h. Following the primary antibody, the samples were incubated with anti-rabbit Alexa 488 secondary antibody and DAPI in PBS containing 0.2% Tween 20 and 3% BSA at 37°C for 1 h. Finally, the samples were mounted in Prolong Gold, and imaged on a Zeiss Axiovert 200M microscope with a Zeiss Axiocam monochrome camera. Images were assembled using Fiji (Schindelin et al., 2012 (link)).

Immediately before live imaging, differentiated myotubes were stained with JF646 HaloTag Ligand (Promega) according to manufacturer specifications. Imaging was performed in Fluorobrite phenol-free culture medium at 37 °C, 5% CO₂ in a stage mounted incubator using a Zeiss LSM 880 AxioObserver microscope with Plan-Apochromat 1.46 NA ×100 oil objective, widefield fluorescence illumination, and Zeiss AxioCam monochrome camera (Fig. 8). For five fast imaging movies, images were captured continuously for 30 s to 2 min with exposure times between 100 and 500 ms. For long imaging movies, images were captured at 1.0 s exposure time with a 15 s time lag over the course of ~50 min.