Objective
Set up a new technique, rat skin IHC, with changes respect 1:
- Change 1: filter secondary Ab
- Change 2: permeability cells with PBS-triton0,3%
Materials
- PBS-Tritón 0,3%
- Goat serum 5% – PBS – Tritón 0,3%
Procedure
- Put the foodpad with OTC on a criomold in the correct orientation and freeze it. Then more OTC in the stage and freeze it together
- Prepare a 24-well plate with PBS
- Cut the tissue with cryostat (60 µm) and put it in wells
- Wash with PBS-triton 0,3% (x2)
- Block: Goat serum (PBS-tritón0,3%+Goat5%) in agitation ~2,5h
- Primary antibody o/n 4ºC in agitation: (1) Neurofilaments 1:2000 (2) Ab-neurofascin 1:500 (3) Ab-CNTN 1:500 (4) MBP 1:500
- 26.02.2014/ Put the plate about 1h at room temperature
- Wash with PBS-tritón 0,3% in agitation 3 times
- Prepare and filter secondary antibody o/n 4ºC in agitation: (1) GAR-594 1:500 (2) GAC-488 1:500 (3) GAM-594 1:500 (4) GAM-594 1:500. Cover the plate with parafilm to avoid dehydration.
- 27.02.2014/ Put the plate about 1h at room temperature
- Wash 4 times with PBS-tritón0,3% in agitation and darkness
- Wash 1 time with PBS
- Put the cuts in slides (1 per condition) in the same direction
- Let dry
- Dehydrate with alcohols: (1st) EtOH50º (2nd)EtOH70º (3rd)EtOH96º (4th)EtOH100º, 10 minutes each
- Mount with DPX
Results & conclusions
(1) Neurofilament: we can see nerves going up and down, so it’s difficult to follow it. Moreover, it’s really positive when the nerve is cut. However, permeability tissue is better to see neurofilaments.
(2) Neurofascin: We cannot see clearly nerves as we see last week
(3) Contactin: Permeability tissue is worst for the background increment because our secondary antibody is anti-mouse (evolutionary close to rat). We expected to see a nerve contactin-positive arriving to epidermal’s entrances (like in paper), but we can’t see it.
(4) MBP: We still see secondary antibody lumps. We see a lot of backgroung like in contactin for the same reason. Also, in this condition we only have the finals cuts, so it’s better to have one foodpad per condition.
(*) We’ll try with nerve because we know how it looks. We’ll do it with free-floating technique.
(*) We’ll do a new IHC: one footpad with antibodies anti-neurofilament and anti-MBP. This way, we will try to find nerves with myelin.
