These samples are between 60-65 dpc. Also, the article uses antibodies that are no longer available through Dako (with exception of the Insulin Ab, which I have). I have a cell signaling Glucagon and Amylase, which neither provide positive signal in the pig; while Insulin has an atypical staining pattern, almost epithelial, at this stage. My Somatostatin provides signal in very few cells. I have no issues with CK7 from Dako, but have had no success in the pig using Anti-Amylase from several suppliers (Abcam, Cell Signaling and Santa Cruz; Rabbit, goat and goat respectively_). I am going to look at my most recent round of IHC today.

My brief protocol for Flash frozen fresh OCT embedded tissues:

4% cold PFA fix at Room Temp for 5 minutes

PBS rinse then 50mM glycine for 5 min

Blocking:

(Dako blocking buffer serum free for 10 min, then in (5% donkey serum, 1% BSA, 0.1% Tx100) for 45 minutes at RT. 

Primary: 1 hour RT in 50% diluted Blocking buffer

Wash 3 x 5 min PBS +0.01% TX100

Secondary: 45 minutes (combination of Jackson Immuno and invitrogen Abs)

Wash 3 x 5 min PBS +0.01% TX100

Prolong Gold + DAPI/Coverslip

Let cure Overnight (but this has not been long enough as of late).

I switched over to my RNA today to see what I can see using RT-PCR, I should know what to expect for protein expression to some extent soon. Severely limited tissue and RNA unfortunately.