The
determination of specific immunoglobulins (Igs) against fecal bacteria
was as previously described,33 (link) with the
following modifications. Removal of debris/rat cells was performed
via filtration through a 100 μm strainer and centrifugation
at 400g for 10 min (4 °C). After being washed
and heat-killed, bacteria were resuspended in 20 mL of PBS, and 100
μL of this suspension was used for overnight coating (4 °C)
in a 96-well ELISA plate. The number of bacteria in each suspension
was measured by spectrophotometry (600 nm) and adjusted across experimental
groups. Blocking was carried out as already described.33 (link) Rat sera were diluted 1:100 and incubated overnight
at 4 °C for detection of IgG, IgM, and IgA. Incubation with secondary
antirat IgG, IgM, and IgA (Bionova, Spain) (1:10,000) for 1.5h in
darkness was followed by the addition of the HRP substrate (Sigma-Aldrich,
USA). Absorbance was measured using a Nanoquant Infinite M200 Pro
multiplate reader (Tecan, Switzerland).
determination of specific immunoglobulins (Igs) against fecal bacteria
was as previously described,33 (link) with the
following modifications. Removal of debris/rat cells was performed
via filtration through a 100 μm strainer and centrifugation
at 400g for 10 min (4 °C). After being washed
and heat-killed, bacteria were resuspended in 20 mL of PBS, and 100
μL of this suspension was used for overnight coating (4 °C)
in a 96-well ELISA plate. The number of bacteria in each suspension
was measured by spectrophotometry (600 nm) and adjusted across experimental
groups. Blocking was carried out as already described.33 (link) Rat sera were diluted 1:100 and incubated overnight
at 4 °C for detection of IgG, IgM, and IgA. Incubation with secondary
antirat IgG, IgM, and IgA (Bionova, Spain) (1:10,000) for 1.5h in
darkness was followed by the addition of the HRP substrate (Sigma-Aldrich,
USA). Absorbance was measured using a Nanoquant Infinite M200 Pro
multiplate reader (Tecan, Switzerland).
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