Paraffin-embedded tissue samples were sectioned at
a thickness of
3–4 μm on charged microscope slides. Samples were deparaffinized
using CitriSolv (Decon Laboratories, Inc., King of Prussia, PA) and
rehydrated sequentially in graded alcohols. Antigen retrieval was
performed by incubating slides in 10 μg/mL proteinase K (MilliporeSigma)
in PK buffer (0.6 M Tris (pH 7.5)/0.1% CaCl2) for 10 min
at RT. Blocking of endogenous peroxidase and alkaline phosphatase
was performed by incubating slides in Rodent Block M (BioCare Medical,
Pacheco, CA) followed by incubation with BLOXALL Endogenous Blocking
Solution (Vector Laboratories, Burlingame, CA). Slides were washed
in TBS buffer and incubated with primary rabbit Salmonella O Antiserum
(Group B Factors 1, 4, 5, 12, #BD 229481, Becton, Dickinson and Company)
for 1 h at 1:5000 dilution. Slides were then incubated in AP-polymer
(Rabbit on Rodent AP-Polymer, BioCare Medical) followed by Vina Green
Chromogen (BioCare Medical, Pacheco, CA) treatment. Tissues were finally
counterstained with hematoxylin (BioCare Medical) before mounting
with EcoMount (BioCare Medical). Slides were cured at 60–70
°C for 15 min.
a thickness of
3–4 μm on charged microscope slides. Samples were deparaffinized
using CitriSolv (Decon Laboratories, Inc., King of Prussia, PA) and
rehydrated sequentially in graded alcohols. Antigen retrieval was
performed by incubating slides in 10 μg/mL proteinase K (MilliporeSigma)
in PK buffer (0.6 M Tris (pH 7.5)/0.1% CaCl2) for 10 min
at RT. Blocking of endogenous peroxidase and alkaline phosphatase
was performed by incubating slides in Rodent Block M (BioCare Medical,
Pacheco, CA) followed by incubation with BLOXALL Endogenous Blocking
Solution (Vector Laboratories, Burlingame, CA). Slides were washed
in TBS buffer and incubated with primary rabbit Salmonella O Antiserum
(Group B Factors 1, 4, 5, 12, #BD 229481, Becton, Dickinson and Company)
for 1 h at 1:5000 dilution. Slides were then incubated in AP-polymer
(Rabbit on Rodent AP-Polymer, BioCare Medical) followed by Vina Green
Chromogen (BioCare Medical, Pacheco, CA) treatment. Tissues were finally
counterstained with hematoxylin (BioCare Medical) before mounting
with EcoMount (BioCare Medical). Slides were cured at 60–70
°C for 15 min.