Edinburgh Core Database Group

Capel Group

Cohn Group

Gaido Group

Jain Group

Keast Group

Lessard Group

Little Group

McMahon Group

Mendelsohn Group

Potter Group

Southard-Smith Group

Vezina Group

 

 

Gaido Group Protocols

Fluorescent Immunocytochemistry Protocol on Frozen sections

  1. Prepare cryostat section at 5-7 mm (for In situ, may 10 mm)
  2. Fix with 4% paraformaldehyde (diluted 16% paraformaldehyde at 1:4 in PBS), 10 min, room temperature
  3. Wash with PBS/Triton, 5 min X 3
  4. Block with Blocking buffer (500 ml normal goat serum to 10 ml PBS + 10 ml Triton, serum match to species the second antibody from), 1 hr, room temperature
  5. Wash with PBS, 5 min X 1
  6. Primary antibody diluted in 3% BSA (300mg in 10 ml PBS), overnight, at 4 °C
  7. Wash with PBS, 5 min X 3
  8. Incubate with biotinylated secondary antibody, 1:500 dilution (pay attention to different species, against primary), 1 hr, room temperature
  9. Wash with PBS, 5 min X 3
  10.  Mount coverslips with VectaMount
  11.  Visualization using microscope
  12. Analysis using Confocal Software

Whole-Mount Indirect Fluorescent Immunohistochemistry – Double or multiple staining

*All incubations and washes are carried out at 4 ºC on an orbital shaker.

  1. Tissue samples are fixed with 4% paraformaldehyde in PBS (1:4, purchased 16%, diluted with PBS), overnight, 4 ºC. (Keep samples in tubes?)
  2. Rinse, 2 times in PBS.
  3. Quench in 50 mM NH4CL, 15 min.
  4. Block in blocking buffer, 24 hr (1% BSA, 0.1% Saponin, 0.02% sodium azide in PBS) [100mg BSA+10ul Saponin+2ul sodium azide ------à10 ml PBS].         You also need to try blocking with Triton X-100 (0.1%) instead of saponin. 
  5. Incubated in FIRST primary antibody in fresh blocking buffer, 24 hr, 4 ºC.       You can incubate all primary antibodies at the same time.
  6. Wash 5 times, 1 hr/each, in blocking buffer.
  7. Incubated in secondary antibody in fresh blocking buffer, 24 hr, 4 ºC.              You can incubate all secondary antibodies at the same time.
  8. Wash 5 times, 1 hr/each, in blocking buffer.

Fluorescently labeled samples are image by laser scanning confocal microscope. Whole-mount samples are viewed in antifade buffer with glycerol in a culture dish with attached coverslip.  For viewing, use the special coverslip glass-bottomed culture dishes without a coverslip. 

Staining of frozen sections for LCM

Follow manufacturer’s protocol for Arcturus HistoGene™ LCM Frozen Section Staining Kit (catalogue # KIT0401).  NOTE:  The only modification to this protocol is the substitution of kit staining solution for Gils 3 hematoxylin.

RNA isolation from LCM samples

Follow manufacturer’s protocol for Arcturus PicoPure RNA isolation kit (catalogue # KIT0204).  Note:  The ‘optional’ DNase treatment is performed on all isolations.

RNA isolation from whole testis

For tissue from gestation days 11-16 follow manufacturer’s protocol for Qiagen RNeasy Micro kit (catalogue # 74004).  For tissue from gestation day 18 and postnatal day 2 follow manufacturer’s protocol for Qiagen RNeasy Mini kis (catalogue # 74104).  NOTE:  The only modification to the Qiagen protocols is as follows:

Homogenize tissue in 500 ul RNA-Stat 60 (TelTest catalogue # CS-111) and perform chloroform extraction as per manufacturer’s instructions.  The resulting supernatant is then treated as the “homogenized lysate” in the Qiagen protocol.  The Qiagen protocol is then followed for the remainder of the extraction.  DNase treatment is performed on all isolations.

RNA amplification and labeling of LCM samples for microarrays

Proposed kit and protocol to be followed is Epicentre TargetAmp 2-Round Aminoallyl-aRNA Amplification kit 1.0 (catalogue # TAA2R4924).  Evaluation of this kit is still in progress.

Microarrays

Follow Affymetrix GeneChip Expression Analysis manual (part # 701021 rev. 4) for cRNA fragmentation, target hybridization, and array washing, staining and scanning.

 

Group Description