Question (68361) | Anti-Hypophosphorylated Neurofilament H antibody [N52] (ab82259)

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Question

Dear Supplier,

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



GENERAL INFORMATION

Antibody Storage Conditions (temperature/reconstitution etc)


-200C

Description of the Problem (high background, low signal, non-specific staining etc)



Low and patchy signal.


Sample (Species/Tissue/Cell Type/Cell Line etc)


Nerve tissue

Fixation of Sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration)



Paraformaldehyde

Antigen Retrieval (Enzymatic method, Heat mediated technique etc)



Enzymatic method




Permeabilization step


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.

Detection Method



Fluorescent labeling

Positive and negative controls used (please specify)



Positive control was used.

OPTIMIZATION ATTEMPTS (PROBLEM SOLVING)




How many times have you tried the IHC?


Several times




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.

Additional Notes



Document attached with image.

Thanks


Kind Regards,

Answer

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

Questions:

- 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:

https://www.abcam.com/index.html?pageconfig=resource&rid=11462

I hope this will be useful for you and for your customer.

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