Antibody validation
This commercial rabbit antibody is an affinity isolated polyclonal antibody raised against a synthetic peptide derived from the C-terminal region of the mouse claudin-15 protein. Our validation included staining of formalin-fixed, paraffin-embedded human duodenum, where the antibody decorated tight junctions throughout the crypt-villus axis and demonstrating increased expression in adults relative to children. The antibody also reacts with mouse claudin-15, and signal was similar in wild type and claudin-2 knockout mice (indicating that the antibody does not cross-react with claudin-2, which also forms a sodium channel).
Immunostaining
Antigen retrieval with Target Retrieval Solution, pH9 (Agilent Technologies, Cat. #S2367) for 40 minutes at 95°C.
Primary and secondary incubations performed used the following reagents:
Antibody | Source | Catalog # | Lot # | RRID # | Working Dilution/Concentration |
| Rabbit anti-claudin-15 antibody | ThermoFisher | QJ228299 | AB_2533391 | 1.5 µg/ml | |
| Mouse anti-E-cadherin antibody [M168] | Abcam | GR216035 | AB_1310159 | 1 µg/ml | |
| Rat anti-actin antibody [MAC 237] | Abcam | ab50591 | GR168034 | AB_867488 | 8 µg/ml |
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| Alexa Fluor 488 AffiniPure F(ab’)2 donkey anti-mouse IgG (H+L) | Jackson Immunoresearch | 118237 | AB_2341099 | 1.5 µg/ml | |
| Alexa Fluor 594 AffiniPure F(ab’)2 donkey anti-rabbit IgG (H+L) | Jackson Immunoresearch | 124065 | AB_2340622 | 1.5 µg/ml | |
| Alexa Fluor 647 AffiniPure F(ab’)2 donkey anti-rat IgG (H+L) | Jackson Immunoresearch | 123491 | AB_2340696 | 1.5 µg/ml | |
| Hoechst 33342 | Life Technologies | H3570 |
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| 10 µg/ml |
c
Duodenum
Brigham and
Women's
Hospital
Harvard
Medical
School
© Copyright JR Turner 2020.