PE Anti-Heme Oxygenase 1 抗体 [HO-1-2] (ab83214)

Thank you for getting in touch.

I am sorry to hear that ab83214 Anti-Heme Oxygenase 1 antibody [HO-1-2] (Phycoerythrin) is not working as expected in your experiment. We have tested ab83214 in flow cytometry and with Mouse, Rat, Guinea pig, Hamster, Dog, Human and Monkey samples, and are happy to provide a suitable resolution for you if it is not working as expected. This includes:


Troubleshooting or protocol optimization steps

A free replacement

Credit note or refund

In answer to your question, I contacted the collaborating laboratories who tested this antibody in flow cytometry, and the provided a protocol which I have copied below. Please note this would be a guideline only and may require some individual optimization. For permeabilization, they used a commercially available flow cytometry permeabilization buffer so I am sorry we don't have further details about what this contains as this information is proprietary. I can suggest to try a commercially avaible buffer or to try for example PBS containing 0.2% saponin. Permeabilize for 10 minutes and also include the saponin in the antibody dilution buffers and wash buffers.


In order to help you if you have further concerns regarding the results, could you please provide the details below along with any images or data that could help me understand the problem.


A brief description of the experiment and protocol you were using (is this a novel or published protocol?), along with how your results differed from those expected

Sample preparation (cell type, species) and number of cells used

Permeabilization and fixation method

Blocking/fixation conditions (post/pre fixation)

Primary antibody conditions (diluent/dilution, incubation time/temperature)

Secondary antibody conditions (diluent/dilution, incubation time/temperature)

Wash steps

Detection and analysis method

Controls used

If you also know the date of purchase and order/lot/PO number, then please include these as well


Thank you in advance. I look forward to hearing from you with the requested information and hope we can resolve this for you as soon as possible.


 

PROTOCOL FROM THE LABORATORY:

Please note this would be a guideline only and may require some individual optimization.


Materials: Maintain Reagents and buffers at 4˚C


Fluorochrome-conjugated primary antibody

Fluorochrome-conjugated isotype control antibody

Fixation/ Permeabilization Buffer

Permeabilization Buffer

PBS (Phosphate Buffered Saline)

FBS (Fetal Bovine Serum)

10 x 75mm Tubes 


Harvest N x 106 cells and wash twice in N x 1 mL of PBS followed by centrifugation at 400 x g for 7 minutes (N=number of samples to be analyzed).  


Re-suspend cells in N x 1 mL of Fixation/ Permeabilization buffer.


Mix well by inverting and incubate for 30 minutes at 4°C.


Mix well by inverting and centrifuge at 400 x g for 7 minutes.


Decant supernatant and re-suspend cells with N x 1 mL of Permeabilization Buffer.


Centrifuge at 400 x g for 7 minutes.


Decant supernatant and re-suspend pellet in N x 50 µL of Permeabilization Buffer.


Gently mix by pipette and aliquot the cell suspension in 50 µL aliquots.


Add the appropriate amount of antibody, to the cell aliquots, to achieve the target working concentration.


Gently mix by pipette and incubate for 30 minutes in the dark at 4°C.


Add 1 mL of Permeabilization Buffer.


Gently mix by pipette and incubate for 5 minutes at 4°C.


Centrifuge at 400 x g for 7 minutes.


Decant supernatant and add 1 mL of Permeabilization Buffer.


Gently mix by pipette and incubate for 5 minutes at 4°C.


Centrifuge at 400 x g for 7 minutes.


Decant supernatant and wash in 1 mL of PBS.


Gently mix by pipette and centrifuge at 400 x g for 7 minutes.


Decant supernatant and wash in 1 mL of PBS.


Gently mix by pipette and centrifuge at 400 x g for 7 minutes.


Decant supernatant and gently re-suspend cell pellet by pipette with 500 µL of PBS containing 1% FBS for evaluation by flow cytometer.