Autosamdri 815 critical point dryer

Fixed specimens for scanning electron microscopy were dehydrated through a graded series of ethanol, CO₂-critical point dried (Autosamdri-815 critical point dryer, Tousimis Research Corporation, MD, USA) and coated with platinum-palladium (JEOL FC-2300 HR sputter coater, JEOL Ltd., Tokyo, Japan). Afterwards, tardigrades were examined and digitally photographed with a JEOL JSM-6335F field emission scanning electron microscope (JEOL Ltd., Tokyo, Japan).

Microstructure characterization with scanning electron microscopy (SEM) was carried out by adapting the protocols in [35 (link),41 (link)]. Briefly, COL1, HC-L-ECM and LC-L-ECM thin structures were fixed for 48 h in 4% PFA in PBS and then washed three times in 0.1 M phosphate buffer (PB) for 10 min. Resulting samples were incubated in 4% osmium tetroxide for 90 min followed by successive washes in deionized water until there was absence of osmium tetroxide. Then, samples were dehydrated in increasing concentrations of ethanol solutions and preserved in absolute ethanol at 4 °C and critical point dried using an autosamdri-815 critical point dryer (Tousimis, Rockville, MD, USA). Samples were mounted using conductive adhesive tabs (TED PELLA, Redding, CA, USA) for imaging and were carbon coated before imaging with a JSM-6510 (JEOL, Tokyo, Japan) scanning electron microscope at 15 kV. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to process the SEM images. Density of fibers per area was obtained by applying a threshold to isolate the fibers in a specific plane to quantify them, while diameter of fibers was estimated as the average of ten randomly-selected fibers in three different zones of each sample.

Formalin-fixed samples were decanted in deionized water for 15 min, dehydrated with increasing grades of ethanol, critical-point-dried with an Autosamdri-815 Critical Point Dryer (Tousimis, Rockville, MD, USA), mounted onto 12.5-mm sample stubs, sputter-coated with platinum (40 mA, 2 × 2.5 min) using an Emitech K575X Sputter Coater (Quorum Technologies, Lewes, UK), and then examined under the FEI Nova NanoSEM 230 field-emission scanning electron microscope (Thermo Fisher Scientific, Waltham, MA, USA). Snapshots of the longitudinal collagen fibres and tendon surfaces were obtained from 100× to 120,000× magnifications.

For wholemount immunofluorescence, HNECs were fixed in −20°C methanol or 4% paraformaldehyde for 10 min as previously described (31 (link)). Transwell filters were cut out of the plastic supports and placed in a humid chamber for staining. Samples were blocked in 10% normal horse serum and 0.1% Triton X-100 in PBS and incubated with primary antibodies for 1–2 h, then with Alexa dye conjugated secondary antibodies (Thermo Fisher) for 30 min at room temperature. Filters were mounted in Mowiol mounting medium containing 2% N-propyl gallate (Sigma). Samples were imaged with a Leica SP8 or Zeiss LSM 900 confocal microscope. For antibodies and fixation conditions, see Supplemental Table S1. For SEM, Transwell filters were fixed in 2% glutaraldehyde, 4% paraformaldehyde in 0.1 M NaCacodylate buffer, pH 7.4 at 4°C overnight. Samples were osmicated, dehydrated, dried with a Tousimis Autosamdri-815 critical point dryer. Samples were mounted luminal side up, sputter coated with 100 Å layer of Au/Pd. Images were acquired with a Hitachi S-3400N VP-SEM microscope operated at 10–15 kV, with a working distance of 7–10 mm and using secondary electron detection.

Unless otherwise stated, all reagents were purchased from Electron Microscopy Sciences (Hatfield, PA, USA) and all specimen preparation was performed at the Electron Microscopy Core Facility, University of Missouri. Midguts were dissected from HWE mosquitoes and fixed in 2% (v/v) paraformaldehyde, 2% (v/v) glutaraldehyde containing 100 mM sodium cacodylate buffer, pH 7.35. Fixed tissues were rinsed with 100 mM sodium cacodylate buffer, pH 7.35 containing 130 mM sucrose and further rinsed with water. Secondary fixation was performed using 1% osmium tetroxide (Ted Pella, Redding, CA, USA) in cacodylate buffer followed by treatment in a Pelco Biowave (Ted Pella) operated at 100 Watts for 1 min. Specimens were next incubated at 4 °C for 1 h, then rinsed with cacodylate buffer and thereafter with distilled water. Using the Pelco Biowave, a graded dehydration series (100 Watts for 40 s per exchange) was performed using ethanol. Samples were dried using the Tousimis Autosamdri 815 critical point dryer (Tousimis, Rockville, MD, USA), and then sputter-coated with 10 nm of platinum using the EMS 150T-ES Sputter Coater. Images were acquired with a FEI Quanta 600F environmental scanning electron microscope (FEI, Hillsboro, OR, USA).