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Please see technical questionnaire attached provided by the customer regarding technical issue with AB82259, Anti-Hypophosphorylated Neurofilament H antibody [NF-200.
This was purchased on our PO# PO-12555, dated 05/12/11.
Customer has been advised that this technical inquiry is well over 6 months Abpromise guarantee. I have also requested for any available images, she will email once she find them.
Antibody Code: AB82259
Batch Number: GR190-9
Antibody Storage Conditions (temperature/reconstitution etc)
Description of the Problem (high background, low signal, non-specific staining etc)
Low and patchy signal.
Sample (Species/Tissue/Cell Type/Cell Line etc)
Fixation of Sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration)
Antigen Retrieval (Enzymatic method, Heat mediated technique etc)
10% triton during the blocking step
Blocking Conditions (Buffer/time period, Blocking agent)
Blocked in normal goat serum for 30mins
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)
Abcam (ab82259) NF200. Dilution = 1:200 in normal goat serum. Incubation time- overnight followed by washing in 0.1M PBS.
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)
Invitrogen alexa fluor 488 goat anti-mouse secondary antibody. Dilution used = 1:1000 in normal goat serum. Incubation time = 2hours. Followed by washing slides in 0.1M PBS.
Positive and negative controls used (please specify)
Positive control was used.
OPTIMIZATION ATTEMPTS (PROBLEM SOLVING)
How many times have you tried the IHC?
Have you run a “No Primary” control? No (Delete one)
Do you obtain the same results every time? No (Delete one)
What steps have you altered?
We have tried using different dilution factors and also with and without the antigen retrieval step. When we used a different primary antibody on the same tissue we obtained a better result.
Document attached with image.
Asked on Dec 03 2012
Thank you for your enquiry regarding ab82259 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.
After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:
1) It would useful to get an image (saved as a jpeg file) from the customer.
2) Sample type: Nerve tissue
- Is the sample from central or peripheral nerve tissue?
- From which species?
3) Antigen retriaval:Enzymatic method can damage the tissue structure of it is not optimised. Has the customer tried HIER and if so for ho w long?
4) Permeabilization: 10% triton
The concentration is extremely high and it may well be the main factor causing the unexpected staining pattern. For immunostaining Tween 20, Saponin, Digitonin and Leucoperm from 0.2 to 0.5%. Customer can find some useful information at this site:
I hope this will be useful for you and for your customer.
Answered on Dec 03 2012