For fixation the following solutions were used: 1. Specimens for control: 5% GA (Serva) buffered with 0.15 M sodium cacodylate, pH 7.4. 2. Specimens for improved contrast with tannic acid: 5% GA buffered with 0.15 M sodium cacodylate, pH 7.4 + 1% tannic acid (Sigma-Aldrich Chemie, München, Germany).
Among these fixation solutions, the ethanol/acetic acid solution performed the best since the W/ H ratios of cells fixed by it was the closest to 1, especially for P. putida and B. subtilis.
After 15 min, 1.5 M glycine was added up to one-tenth the volume of the fixation solution to stop the cross-linking.
Shortly, 4 days after infection, the chips were fixed using the Fixation Solution provided with the kit for 1 h at 25 C, washed with PBS and permeabilised with the Permeabilisation Solution from the kit for 2 minutes on ice.
The BAL samples were again centrifuged at 300 g for 10 minutes, then the supernatant was removed carefully, the fixation solution 1x added slowly and the samples kept at 4 8°C.
The contra-lateral tibiae were prepared for histomorphometrical examination: After explantation, the proximal and the distal epiphysis were removed to enable the fixation solution (10% normal buffered formaldehyde) to infiltrate into the whole specimen.
The brains were extracted from the skull and postfixed in the fixation solution for 12h, cryoprotected overnight in 30% sucrose, and then sectioned in coronal plane at a 40 μm thickness using a cryostat (Leica Microsystems, Germany).
Silver staining was accessed by immersing the 2-DE gel in the fixation solution containing methanol (50%), acetic acid (12%), and water (38%) for 30 minutes, followed by 50% ethanol for 60 minutes.
Turn off the power supply and carefully open the sandwich gel, and place it into the fixation solution, at least 1 hour.
Brains were removed and postfixed in the fixation solution overnight and stored in a 1 10 dilution of the same solution in 0.1 M PB.
