As indicated, excised vaginas were placed in formaldehyde and paraffin embedded and 10μm tissue sections were mounted on glass slides and de-paraffinized by sequential immersion in 100% xylene, 100% ethanol, 96% ethanol, and sterile DEPC-treated water. Antigen retrieval was performed using 10mM sodium citrate buffer (pH 6.0) containing 0.05% Tween 20 (both Sigma-Aldrich) (20 minutes at 95°C). After 3 PBS washes, sections were incubated overnight with 10% normal donkey serum (Abcam, Cambridge MA) at 4°C, incubated 1 h at ambient temperate with rabbit anti-desmoglein-1 (clone EPR6766(B)), washed, and incubated 1 h with AlexaFluor® 488-labeled donkey anti-rabbit IgG labeled (both antibodies Abcam) (all antibodies were diluted in PBS with 1% BSA and 0.05% Tween 20). Sections were stained with DAPI, and evaluated by confocal microscopy for relative expression of DSG1a protein (defined by calculating pixel numbers per 100 μm2) using ImageJ software53 (link). To evaluate relative DSG1a protein expression, images were acquired using identical parameters and their contrast altered equally (investigators performing such measurements were blinded to study group designation).
Alexafluor 488 labeled donkey anti rabbit igg labeled
Manufactured by Abcam
AlexaFluor® 488-labeled donkey anti-rabbit IgG is a secondary antibody conjugated with the AlexaFluor® 488 fluorescent dye. It is designed to detect and bind to rabbit primary antibodies.
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2 protocols using alexafluor 488 labeled donkey anti rabbit igg labeled
1
Immunofluorescent Detection of Desmoglein-1
2
Immunofluorescent Detection of Desmoglein-1
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As indicated, excised vaginas were placed in formaldehyde and paraffin embedded and 10μm tissue sections were mounted on glass slides and de-paraffinized by sequential immersion in 100% xylene, 100% ethanol, 96% ethanol, and sterile DEPC-treated water. Antigen retrieval was performed using 10mM sodium citrate buffer (pH 6.0) containing 0.05% Tween 20 (both Sigma-Aldrich) (20 minutes at 95°C). After 3 PBS washes, sections were incubated overnight with 10% normal donkey serum (Abcam, Cambridge MA) at 4°C, incubated 1 h at ambient temperate with rabbit anti-desmoglein-1 (clone EPR6766(B)), washed, and incubated 1 h with AlexaFluor® 488-labeled donkey anti-rabbit IgG labeled (both antibodies Abcam) (all antibodies were diluted in PBS with 1% BSA and 0.05% Tween 20). Sections were stained with DAPI, and evaluated by confocal microscopy for relative expression of DSG1a protein (defined by calculating pixel numbers per 100 μm2) using ImageJ software53 (link). To evaluate relative DSG1a protein expression, images were acquired using identical parameters and their contrast altered equally (investigators performing such measurements were blinded to study group designation).
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