Samdri 795 critical point drier

S. aureus Newman Δspa was grown in phenol-free RPMI medium (Gibco) at 37 °C overnight. The bacteria were washed two times with PBS and resuspended in 0.05% bovine serum albumin (BSA) in phenol-free RPMI to an OD₆₀₀ ~ 1. 500 μl of bacteria was mixed with 500 μl of NHS (BioIVT) or 500 μl 0.05% BSA in phenol-free RPMI. The samples were incubated at 37 °C for 8 h with shaking. The bacteria were then washed three times with PBS. For EM analyses, samples were fixed 1:1 with a 2x fixative [5% glutaraldehyde, 4% paraformaldehyde (PFA) in 0.2 M sodium cacodylate buffer] and dehydrated through a graded series of ethanol. Samples were critical point dried using liquid carbon dioxide in a Tousimis Samdri 795 Critical Point Drier, coated with carbon in a Quorum EMS 150T ES (Quorum Technologies Ltd) and examined in a Zeiss Supra Field Emission Scanning Electron Microscope (Carl Zeiss Microscopy), using an accelerating voltage of 8 KV.

Tracheas from E18.5 WT or Tnrc6a^gt/gt embryonic lungs were dissected, fixed in 2.5% glutaraldehyde, 0.1 M sodium Cacodylate, 0.2 M Sucrose, 5 mM MgCl2 pH 7.4 and dehydrated through a graded series of ethanol dilutions. Samples were processed using liquid carbon dioxide in a Tousimis Samdri 795 Critical Point Drier (Rockville MD), sputter coated with chromium in a Quorum EMS 150 T ES (Quorum Technologies Ltd, United Kingdom) and examined in a Zeiss Supra Field Emission Scanning Electron Microscope (Carl Zeiss Microscopy, LLC North America), using an accelerating voltage of 2 kV. For quantitative analysis, lungs (n = 2) were examined and about 120 cells were counted for each sample.