Antibody validation
This commercial rabbit antibody from Atlas Antibodies (HPA043207) is a polyclonal affinity isolated antibody raised agasint the sequence MVRLVLPNPGLDARIPSLAELETIEQEEASSRPKWDNK. Enhanced validation by the manufacturer was by comparison of IHC stains to RNASeq data. Our validation included staining of formalin-fixed, paraffin-embedded human duodenum, where SLC6A19 is detected within the apical brush border of villous, but not crypt, epithelial cells (right, upper image). In contrast, expression is not detected in the colon (right, lower image), as expected.
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-B0AT1 antibody | Atlas Antibodies |
R41029 |
AB_2678363 |
3 µg/ml |
|
| Mouse anti-E Cadherin antibody [M168] | Abcam |
GR21603-5 |
AB_1310159 |
1:300 |
|
| Rat anti-ZO-1 [1F2G12] | Turner lab |
|
|
AB_2783875 |
1:50 |
|
|
|
|
|
||
|
|
|
|
|
||
| Alexa Fluor® 488 AffiniPure F(ab’)2 Donkey Anti-Mouse IgG (H+L) | Jackson Immunoresearch |
715-546-151 |
130065 |
AB_2341099 |
3 µg/ml |
| Alexa Fluor® 594 AffiniPure F(ab’)₂ Fragment Donkey Anti-Rabbit IgG (H+L) | Jackson Immunoresearch |
711-586-152 |
114063 |
AB_2340622 |
3 µg/ml |
| Alexa Fluor® 647 AffiniPure F(ab’)₂ Fragment Donkey Anti-Rat IgG (H+L) | Jackson Immunoresearch |
712-606-153 |
123491 |
AB_2340696 |
3 µg/ml |
| Hoechst 33342 | Life Technologies |
H3570 |
|
|
10 µg/ml |

Duodenum

Colon
Jerrold R. Turner, MD, PhD
Professor of Pathology and Medicine
Brigham and
Women's
Hospital
Harvard
Medical
School
TH 613
20 Shattuck St.
Boston, MA 02115.
© Copyright JR Turner 2020.