Cryopreserved free-floating tissue sections were rinsed with 0.1M PBS (pH 7.6) (5 times (5 minutes each time), blocked in 4% normal donkey serum (4% NDS) and 0.3% PBS-TX for 60 minutes, and incubated in primary antisera including GFP (chicken; Abcam, RRID: AB300798, 1:1500), tyrosine hydroxylase (TH; rabbit; Millipore AB152, RRID: AB390204, 1:500), oxytocin (OT; rabbit; Peninsula Labs; RRID:AB_518522; 1:500), and c-Fos (goat; Santa Cruz sc-52-G, RRID:AB_2629503) at room temperature overnight. The following day, tissue sections were rinsed with 0.1M PBS (pH 7.6) 3 times (5 minutes each time), incubated in various secondary antisera [(anti-rabbit alexa 594 (1:500, Invitrogen); anti-rabbit alexa 350 (1:250, Invitrogen), anti-chicken alexa 488 (1:1000, Jackson Labs), anti-goat alexa 594 (1:250, Invitrogen)] in 4% NDS and 0.3% PBS-TX for 2.5 hours. Sections were mounted onto gel-coated slides and coverslipped with antifade mounting medium (VECTASHIELD® HardSet™, Vector Laboratories). Antibody characterizations for GFP, TH, OT, and c-Fos were previously described.29 (link),33 (link),35 (link),37 (link)
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Multimodal Immunofluorescence Labeling of Cryopreserved Tissue
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Other organizations : University at Albany, State University of New York, Albany Medical Center Hospital, Brown Foundation
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Variable analysis
independent variables
- Cryopreserved free-floating tissue sections
- Expression of GFP
- Expression of tyrosine hydroxylase (TH)
- Expression of oxytocin (OT)
- Expression of c-Fos
- 0.1M PBS (pH 7.6) for rinsing
- 4% normal donkey serum (4% NDS) and 0.3% PBS-TX for blocking
- Room temperature for incubation
- Overnight incubation in primary antisera
- 3 rinses with 0.1M PBS (pH 7.6) for 5 minutes each
- 2.5 hours incubation in secondary antisera
- Mounting on gel-coated slides
- Coverslipping with antifade mounting medium
- Antibody characterizations for GFP, TH, OT, and c-Fos were previously described in the referenced studies.
- No negative controls were explicitly mentioned in the protocol.
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