Zinc formaldehyde

For histological analysis, the samples were placed in zinc-formaldehyde (Sigma Aldrich, Taufkirchen, Germany), embedded in paraffin, and cut into 7 µm sections. Standard hematoxylin and eosin (H&E, Carl Roth, Karlsruhe, Germany) and immunofluorescence staining were performed.
Histological severity scoring was performed in a double-blinded manner using the following criteria: 0—unaffected co-culture, 1—mild injury with minor mesothelial loosening, 2—moderate injury with some mesothelial disruption, 3—severe injury with continuous mesothelial disruption and some detachment, and 4—extensive injury, massive mesothelial disruption and detachment.
For immunofluorescence staining, sections were stained as previously described [25 (link)] with primary antibodies against cellular Jun (c-Jun) (Cell Signaling, Danvers, MA, USA) diluted in blocking buffer (1:100), vascular endothelial cadherin (VE-cadherin) (1:250), as well as zonula occludens-1 (ZO-1) (1:1000) (Proteintech, Rosemont, IL, USA). After three washes in tris-buffered saline (TBS) for 10 min, slides were incubated with goat-anti rabbit Alexa Fluor-labeled secondary antibodies (Cell Signaling, Danvers, MA, USA) diluted in blocking buffer.

Mice were sacrificed by isoflurane overdose, and the wound chambers were aseptically removed.
The wound tissue was divided in one half and two quarters for histology, FISH, and CFU analysis. The quarter to be used for CFU analysis was transferred to 1 ml PBS (pH 7.4) and vortexed for 1 minute to homogenize the biofilm. The bacterial suspension was diluted, 100 µl of each dilution plated on Mueller Hinton agar, and plates were incubated over night at 37°C. Colonies were then counted and final counts were calculated taking the dilution factor into account. For histology, the tissue was placed in zinc-formaldehyde (Sigma Aldrich, Taufkirchen, Germany) and embedded in paraffin, cut in 7 µm sections. Following histochemical dyes can be run according to manufactures’ protocols, depending on the analysis requested.
For FISH analysis, tissue was placed in FISH fixation solution (FISHopt^®, MoKi Analytics, Berlin, Germany), embedded in methacylate and processed as published previously [18 (link)], [19 (link)].