Double immunofluorescent staining using two unconjugated primary antisera raised in the same species
Monoclonal antibodies (MAbs) capable of recognizing developmental stage-specific neuronal epitopes are becoming increasingly available. Because most of these MAbs are raised in a single species (mouse), simultaneous immunofluorescent detection of multiple epitopes has been difficult. We have taken a...
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Published in | The journal of histochemistry and cytochemistry Vol. 44; no. 11; pp. 1331 - 1335 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
United States
Histochemical Soc
01.11.1996
SAGE Publications |
Subjects | |
Online Access | Get full text |
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Summary: | Monoclonal antibodies (MAbs) capable of recognizing developmental stage-specific
neuronal epitopes are becoming increasingly available. Because most of these MAbs are
raised in a single species (mouse), simultaneous immunofluorescent detection of
multiple epitopes has been difficult. We have taken advantage of the high sensitivity
of tyramide signal amplification to develop a protocol that permits simultaneous
detection of two antibodies raised in the same species. One primary antibody was
applied at a concentration below the detection limit of fluorescently labeled
secondary antibodies, yet sufficient for detection with the tyramide system. This
first primary antibody was then effectively neglected during application of a second
primary antibody that was detected by conventional fluorescently labeled secondary
antibodies. Specifically, dual labeling for nestin and MAP2 was used to distinguish
neuronal stem cells and precursor cells from immature postmitotic neurons, and
synapsin I and GAP43 immunostaining was used to distinguish neurons with established
synaptic connections from developing neurons. We have used this technique for
staining both tissue sections and cultured cells from the embryonic mouse brain. This
technique should be widely applicable and offers a simple procedure for
simultaneously detecting two antigens when antibodies from only a single species are
available. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0022-1554 1551-5044 |
DOI: | 10.1177/44.11.8918908 |