Rabbit Anti-Armenian hamster IgG H&L (HRP) (ab5745)

Thanks for your message and for kindly providing this further information.

I have copied below the IHC-P testing protocol for this antibody, which includes the antigen retrieval details. I hope this will be helpful. Please note this protocol will be a guideline only and may require some individual optimization.

Please do not hesitate to let me know how you get on with the next experiments. If the experiment is still not working, please do not hesitate to contact me with the following details as previously requested:

1. Please provide the order number and date of purchase.

2. Time for antigen retrieval with paraffin embedded samples will require some individual optimization depending on the individual experiment (sample type, epitope being detected, species etc). Try different time points to see which provides better results, for example 2, 5, 10 and 20 minutes.

3. TCR gamma /TCR delta is a membrane protein. Therefore permeabilization will not be required. Triton is quite a strong detergent and can disrupt membrane proteins and significantly affect the staining.

Try including a more gentle detergent, 0.2% Tween in the wash buffer and antibody dilution buffer. This will help to keep the antibody solubilised and wash away any excess antibody.

I look forward to hearing from you.

Tissue Preparation:
Formalin fixation and embedding in paraffin wax

Tissue Sectioning:
Make 4-um sections and place on pre-cleaned and charged microscope slides. Heat in a tissue-drying oven for 45 minutes at 60°C.

Deparaffinization:
Wash dry slides in 3 changes of xylene – 5 minutes each @ RT

Rehydration:
Wash slides in 3 changes of 100% alcohol – 3 minutes each @ RT
Wash slides in 2 changes of 95% alcohol – 3 minutes each @ RT
Wash slides in 1 change of 80% alcohol – 3 minutes @ RT
Rinse slides in gentle running distilled water – 5 minutes @ RT

Antigen retrieval:
Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C - 20 minutes
Remove from heat and let stand at room temperature in buffer - 20 minutes
Rinse in 1X TBS with Tween (TBST) – 1 minute @ RT

Immunostaining: (Do not allow tissues to dry at any time during the staining procedure)

Apply a universal protein block – 20 minutes @ RT
Drain protein block from slides, apply diluted primary antibody – 45 minutes @ RT
Rinse slides in 1X TBST - 1 minute @ RT
Apply a biotinylated anti-rabbit IgG (H+L) secondary – 30 minutes @ RT
Rinse slides in 1X TBST - 1 minute @ RT
Apply alkaline phosphatase streptavidin – 30 minutes @ RT
Rinse slides in 1X TBST - 1 minute @ RT
Apply alkaline phosphatase chromogen substrate – 30 minutes @ RT
Wash slides in distilled water – 1 minute @ RT

Dehydrate: (This method should only be used if the chromogen substrate is alcohol insoluble (e.g. Vector Red, DAB)

Wash slides in 2 changes of 80% alcohol – 1 minute each @ RT
Wash slides in 2 changes of 95% alcohol – 1 minute each @ RT
Wash slides in 3 changes of 100% alcohol – 1 minute each @ RT
Wash slides in 3 changes of xylene – 1 minute each @ RT
Apply coverslip

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab118864 is tested and covered by our 6 month guarantee for use in IHC-Fr and mouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Although the other antibody has worked well using this procedure, individual antibodies will often require optimization. Protocols are provided as a guideline only and individual optimization will often be required for individual experiments. I would like to offer some suggestions to help optimize the results from ab118864. I would also appreciate if you can confirm some details of the procedure.

1. Please provide the order number and date of purchase.

2. Fixation: for frozen sections, we would recommend to fix in 4% PFA for 10 minutes only. This should mean that antigen retrieval is then not required. Could you confirm these are frozen samples? There is no mention of paraffin embedding.

3. Antigen retrieval: Antigen retrieval is not usually required for frozen sections.

Time for antigen retrieval with paraffin embedded samples will require some individual optimization depending on the individual experiment (sample type, epitope being detected, species etc). Try different time points to see which provides better results, for example 2, 5, 10 and 20 minutes.

4. TCR gamma /TCR delta is a membrane protein. Therefore permeabilization will not be required. Triton is quite a strong detergent and can disrupt membrane proteins and significantly affect the staining.

Try including a more gentle detergent, 0.2% Tween in the wash buffer and antibody dilution buffer. This will help to keep the antibody solubilised and wash away any excess antibody.

5. Please confirm what dilutions of primary antibody have been tried? I can suggest to increase the concentration to increase the signal obtained.

6. Is the secondary antibody working well with other primary antibodies? This will help to assess where the problem is.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details. I hope we can resolve this case for you as soon as possible.

Vielen Dank für Ihre Anrufe.
Wie besprochen können wir den MCP1 Antikörper für Western blot und Durchflußzytometrie garantieren (www.abcam.com/Abpromise). In der Immuncytochemie/Immunfluoreszenz (ICC/IF) wurde er noch nicht getestet, aber es bestehen gute Chancen, dass er funktioniert, da er ja für die Immunhistochemie getestet ist.
DISCOUNT CODE: x
Ablaufdatum: ddmm.2012
Es freut mich zu hören, dass Sie unser Angebot unseren Antikörper Ab21396 in der ICC/IF zu testen, annehmen möchten. Wie am Telefon besprochen, können Sie den Gutscheincode zum Kauf eines weiteren Antikörpers einsetzen, sobald Sie uns ein Abreview für diesen Antikörper eingereicht haben. Der Discountcode hat die Nummer 100%ABR-T7ATQ und kann zum Kauf weiterer Produkte eingesetzt werden.
Der Code wird gültig, sobald Sie uns ein Abreview für diesen Antikörper über den Test in ICC/IF eingereicht haben. Bitte geben Sie diesen Code auch im Abschnitt “Additional Notes” des Abreviews mit an, so dass wir wissen, dass sich dieses Abreview auf die Gutscheinaktion bezieht. Der Code wird dadurch aktiviert. Bitte kontaktieren Sie bei der nächsten Bestellung unsere Kundendienst mit dem Code und der ursprünglichen Bestellnummer.
Die sekundären anti-Armenian Hamster Antikörper, die wir haben sind:
ab5738 (unkonjugiert):
https://www.abcam.com/Goat-Armenian-hamster-IgG-HL-ab5738.html https://www.abcam.com/Goat-Armenian-hamster-IgG-HL-ab5738.html.
ab5744 (biotin):
https://www.abcam.com/Goat-Armenian-hamster-IgG-HL-Biotin-ab5744.html https://www.abcam.com/Goat-Armenian-hamster-IgG-HL-Biotin-ab5744.html.
ab5745 (HRP):
https://www.abcam.com/Rabbit-Armenian-hamster-IgG-HL-HRP-ab5745.html https://www.abcam.com/Rabbit-Armenian-hamster-IgG-HL-HRP-ab5745.html.
ab5746 (AP) :
https://www.abcam.com/Goat-Armenian-hamster-IgG-HL-Alkaline-Phosphatase-ab5746.html https://www.abcam.com/Goat-Armenian-hamster-IgG-HL-Alkaline-Phosphatase-ab5746.html.
Falls Sie an Fluoreszenz-Konjugaten interessiert sind, sind unsere Easy Link-Kits vielleicht etwas für Sie: damit kann jeder Antikörper schnell und einfach mit einem Fluorochrom konjugiert werden. Einzelheiten finden Sie unter: www.abcam.com/Easylink
Bitte zögern Sie nicht, sich wieder an uns zu wenden, falls Sie weitere Fragen haben. Wir freuen uns auf Ihr Abreview, egal ob Ihre Ergebnisse positiv oder negativ sind und wünschen Ihnen viel Glück für Ihre Forschung.
Die Bedingungen dieses Angebotes befinden sich unter dem folgenden Link: www.abcam.com/collaborationdiscount
PS: Die Bestellung (#1064638) wurde wie gewünscht storniert.

Thank you for your enquiry. This product ab36772 was created by immunizing Armenian hamsters with a synthetic peptide conjugated to KLH. The resulting splenocytes were then fused to the tumor cell line SP2/0. We recommend using any HRP conjugated, anti-hamster Ig secondary to detect this product. by Western blot. Also, a secondary antibody that is specifically against Armenian hamster, like ab5745 should be a very good secondary too. If there is anything else that I can help you with, please do not hesitate to contact me.