Table 3. Details of immunofluorescence staining protocol.
Protocol steps | Reagent | Concentration
/dilution |
Time | Catalogue
Number |
---|---|---|---|---|
• Cryopreserved Rat muscles
• Cryostat sections (3μm) |
||||
Fixation | • Paraformaldehyde
• Methanol |
1%
100% |
30 min at RT
5 min at –20°C |
|
Staining | Primary Abs:
MAbs: • anti-α-SKA (10D2) • anti-α-CAA (22D3) • anti-α-SMA (1A4) For details, see Table 1 • anti-vimentin (clone V9) Immunogen: Purified vimentin from porcine eye lens Specificity: the antibody cross-reacts with vimentin in man, cow, dog, hamster, horse, rhesus and African green monkey, rabbit, rat and rat kangaroo. No cross-reaction with mouse vimentin could be demonstrated, and results on chicken specimens are contradictory. Rabbit polyclonal Ab: • anti-α-CAA ( Clément et al., 2003) |
1:50 1:50 1:50 1:300 1:100 |
2 h |
Home made Home made Home made Dako #M 0725 Home made |
Secondary Abs (Jackson Immmunoresearch):
• AffiniPure Goat Anti-Mouse IgG1, Alexa 488 • AffiniPure Goat Anti-Mouse IgG1, Alexa 594 • AffiniPure Goat Anti-Mouse IgG2a, Alexa 488 • AffiniPure Goat Anti-Mouse IgG2a, Alexa 594 • AffiniPure Goat Anti-Rabbit-Alexa 594 |
1:200 1:200 1:200 1:200 1:100 |
1 h |
115-545-205 115-585-205 115-545-206 115-585-206 111-585-144 |