Recombinant Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

This antibody would not distinguish between the mouse M or P lysozyme isoforms as the immunogen sequence in conserved in both.

Thank you for providing that information.

I have had a look at the two rabbit monoclonal against Lysozyme (ab91653 and ab108508). Both of these antibodies should be able to detect all three forms of the protein (the wild type as well as your two mutants). The immunogen used to raise the ab108508 antibody has been taken from a region N-terminal of your mutations, whilst the immunogen used to raise the antibody ab91653 is from the C-terminal domain. Neither immunogen overlaps any of the mutation sites.

I hope this information has beenof help. If you have any further questions please do not hesitate to ask.

Thank you for contacting us yesterday.

I have received further information from the lab regarding the IHC protocol for ab108508. I can
confirm that 0.01M Sodium Citrate Buffer, pH 6.0 was used for antigen retrieval. Here is a copy
of the protocol:

Immunohistochemistry Protocol for Paraffin-embedded Tissues

1. Solutions and reagents
1.1. Xylene
1.2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 70%, 50%)
1.3. Washing buffer/TBST: 1X TBS/0.1% Tween-20, pH to 7.6.
1.4. Distilled water (dH2O)
1.5. Antigen Retrieval Solution: 0.01M Sodium Citrate Buffer, pH 6.0
To prepare Antigen Retrieval stock solutions:
10X Stock: Dissolve 29.4 g sodium citrate trisodium salt dehydrate (C 6H 5Na 3O 7 2H2O in 1 liter of dH2O. Add 5mL Tween-20.
1X Working Solution: Mix 200mL 10X stock with 1800mL dH2O; pH to 6.0
1.6. 3% Hydrogen Peroxide
1.7. Blocking Buffer: 10% serum in PBS (serum origin depends on the host of the secondary antibody)
1.8. Primary Antibody Diluent: 5% serum in PBS (serum origin depends on the host of the
Secondary antibody)
1.9. Hematoxylin
1.10. Permanent Mounting Medium

2. Protocol
2.1. Deparaffinization/Rehydration
2.1.1. Heat slides in an oven at 65 °C for 1 hour.
2.1.2. De-paraffinize/hydrate using the following series of washes: two Xylene washes (3 min each), followed by two 100% ethanol rinses (3 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by TBST wash for 3 min on a shaker.

2.2. Antigen Retrieval
This is recommended Heat Induced Epitope Retrieval (HIER) using Decloaking
Chamber/Pressure Cooker. Hot water bath or Microwave with temperature sensor can be also used
(protocol would vary depending on the method used).
2.2.1. Add 500 ml of dH 2O to Decloaker/Pressure Cooker.
2.2.2. Immerse slides into staining dish containing Antigen Retrieval Solution. Place staining dish into decloaking chamber.
2.2.3. Program to run for 30 seconds at 125° C, followed by 10 seconds at 90° C.
2.2.4. Let it cool down to room temperature (10 – 20 minutes).
2.2.5. Removes slides and rinse in TBST.
2.2.6. Proceed to Staining step.

2.3. Staining
2.3.1. Wash slides with TBST for 3 min on a shaker.
2.3.2. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 5 min.
2.3.3. Wash slides three times with TBST (3 min each on a shaker).
2.3.4. Block slides with the blocking solution for 1 hour.
2.3.5. Dilute primary antibody in primary antibody diluent per recommendation on data sheet.
2.3.6. Apply primary antibody to each section and incubate overnight in the humidified chamber (4 ºC).
2.3.7. Wash slides three times with TBST (3 min each on a shaker).
2.3.8. Apply to each section secondary HRP-conjugated anti-rabbit antibody diluted in the blocking solution per manufacturer’s recommendation; incubate for 30 min at room temperature.
2.3.9. Wash slides three times with TBST (5 min each on a shaker).
2.3.10. Add freshly prepared DAB substrate to the sections and incubate until stain develops (generally 1 min).
2.3.11. Rinse sections with water.
2.3.12. Counterstain with Hematoxylin (generally 10 seconds).
2.3.13. Rinse sections with water.
2.3.14. Dehydrate samples using two washes with 100% Ethanol (3 min each), followed by
two rinses with Xylene (3 min each).
2.3.15. Mount coverslips on slides using permanent mounting medium.

I hope this information helps and that the antibody works well. Please do not hesitate to contact us should you have further problems.

Thank you for contacting us. Based on sequence analysis, immunogen of ab91653 does not share any significant sequence similarity with chicken. For the immunogen of ab108508, it shares 67% identity with chicken Lysozyme C, so it should also be human specific. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.