Transverse sections through the mid-thoracic to lower abdominal/inguinal region of P0, P3 and P7 pups were fixed in 4% paraformaldehyde for 24h and maintained in 50% ethanol solution. Paraffin embedding, perinatal tissue sectioning and H&E staining was performed by the Molecular Pathology Core Facility at UTSW. Brightfield and fluorescent images were acquired using a Keyence BZ-X710 microscope. Indirect immunofluorescence was performed as previously described 21 (link). Primary antibodies used for immunofluorescence include: guinea pig anti-PERILIPIN 1:1000 (Fitzgerald #20R-PP004), chicken anti-GFP 1:500 (Abcam, ab13970), rabbit anti-MESOTHELIN 1:1000 (LSBio #LS-C407883), rabbit anti-MAC-2 1:500 (Cedarlane #CL8942AP), rabbit anti-EFHD1 1:100 (Sigma #SAB3500392), mouse anti EFHD1 1:100 (Abnova #H00080303-M05), mouse anti-AGT 1:100 (Santa Cruz Biotechnology #sc-374511), rat anti-ENDOMUCIN 1:100 (Santa Cruz Biotechnology #sc-65495).
Protocol detail
Perinatal Tissue Immunofluorescence Protocol
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Abdominal
Antibodies
Chicken
Endomucin
Ethanol
Guinea pig
Immunofluorescence
Indirect immunofluorescence
Inguinal region
Mac 1
Mesothelin
Microscope
Mouse
Paraformaldehyde
Perilipin 1
Rabbit
Tissue
Corresponding Organization : The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- Age of pups (P0, P3, P7)
- Morphological changes in the mid-thoracic to lower abdominal/inguinal region as observed through histological analysis (H&E staining)
- Protein expression patterns of PERILIPIN, GFP, MESOTHELIN, MAC-2, EFHD1, AGT, and ENDOMUCIN as observed through immunofluorescence
- Tissue fixation in 4% paraformaldehyde for 24h
- Maintenance in 50% ethanol solution
- Paraffin embedding, perinatal tissue sectioning, and H&E staining performed by the Molecular Pathology Core Facility at UTSW
- Indirect immunofluorescence protocol as previously described
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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