When gels were recovered from mice, they were processed as previously described for paraffin embedding33 (link). Briefly, gels were fixed in 4% neutral buffered formalin overnight and subsequently dehydrated and paraffin embedded. Sections of 4 µm were cut and processed for either hematoxylin eosin staining or for immunofluorescence staining.
After dewaxing, sections were placed in Tris–HCl buffered saline supplemented with Tween-20 (TBS-Tween, 0.1 M Tris HCl, 0.14 M NaCl, 0.05% Tween-20, pH7.6, all reagents from Sigma-Aldrich) for 10 min, then incubated at room temperature for 1 h in TBS-Tween supplemented with 10% goat serum (Sigma-Aldrich) and then for 1 h at room temperature with the primary antibody. Sections were then washed 3 times with TBS-Tween, the secondary antibody applied and incubated for another hour. Sections were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and then mounted.
As primary antibody, human cytokeratin 14 (CK14, 1:500 dilution, polyclonal AF-64, Covance, Princeton, NJ, USA) was used, while as secondary antibody, AlexaFluor 488-labelled goat anti-rabbit IgG was used (1:500, Invitrogen, Thermo Fisher Scientific).
After dewaxing, sections were placed in Tris–HCl buffered saline supplemented with Tween-20 (TBS-Tween, 0.1 M Tris HCl, 0.14 M NaCl, 0.05% Tween-20, pH7.6, all reagents from Sigma-Aldrich) for 10 min, then incubated at room temperature for 1 h in TBS-Tween supplemented with 10% goat serum (Sigma-Aldrich) and then for 1 h at room temperature with the primary antibody. Sections were then washed 3 times with TBS-Tween, the secondary antibody applied and incubated for another hour. Sections were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and then mounted.
As primary antibody, human cytokeratin 14 (CK14, 1:500 dilution, polyclonal AF-64, Covance, Princeton, NJ, USA) was used, while as secondary antibody, AlexaFluor 488-labelled goat anti-rabbit IgG was used (1:500, Invitrogen, Thermo Fisher Scientific).