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First issue: thank you for the offer: we would indeed appreciate a lot to receive a new vial of ab110363 and a purification kit for tissue culture supernatant ab109207 to replace the lost material. This could help us a lot.
Second issue: Questionnaire for ab21682:
It will take approximately 5 minutes to complete the questionnaire.
Please fill-in all fields so that we can better assist you
1) Abcam product code ab21682, rabbit anti-DLK1
2) Abcam order reference number or product batch number : GR31847-1
3) Description of the problem: high aspecific background, impossible/difficult to pick up the DLK1 signal. Conclusion: antibody is not specific, but gives a very particular intracellular/perinuclear staining pattern (=golgi/ER?)
4) Sample preparation:
Species: rat pancreatic beta cells
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other:
Sample preparation : fixation in formol
Positive control: unstimulated cells (MIX1), negative for DLK1
Negative control : cells treated with specific compound to trigger DLK1 expression (MIX2), confirmed at level of qPCR (80.000 fold increase at RNA level), ICC with 5 other rabbit anti DLK1 antibodies, including ab110636
5) Fixation step
If yes: Fixative agent and concentration : formaldehyde 3.7% in PBS
Fixation time: 30 minutes to 1 day
Fixation temperature: cold room 4°C
6) Antigen retrieval method: none, not required for the other tested antibodies, which all were very specific (see picture)
7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?
Permeabilizing agent and concentration:
TritonX100, 0.5% in PBS
8) Blocking agent (eg BSA, serum…): BSA 3%
Blocking time: 30 to 60 minutes
Blocking temperature room temperature
9) Endogenous peroxidases blocked? No, not relevant
Endogenous biotins blocked? No, not relevant
10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1/250, 1/1000
Diluent buffer: PBS
Incubation time: overnight, 4°C
11) Secondary antibody:
Species: donkey anti rabbit
Concentration or dilution: 1/3000
Diluent buffer PBS
Incubation time: 30 minutes
Fluorochrome or enzyme conjugate: 488
12) Washing after primary and secondary antibodies:
Buffer: PBS+3% BSA
Number of washes : 3x 5 minutes
13) Detection method: 488 fluorescence microscope – DAPI for nuclei
14) How many times have you run this staining? 3x, in parallel with other DLK1 antibodies (ab110636, santacruz and an non commercial ab)
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? Not, other antibodies have been used as possitve control, we have no need to optimize this ab further.
Document attachment: see enclosed picture
Asked on Jun 22 2012
Thank you for these precisions.
We are still working on your complaint for ab21682 (anti-DLK1) and we will come back to you as soon as possible.
As requested, I have issued a free of charge replacement with the order number *******.
To check the status of the order please contact our Customer Service team and reference this number.
Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.
Answered on Jun 22 2012