Anti-CD105 antibody [105C02] (ab44967)

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab49679.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Thank you for contacting Abcam.
Earlier today we discussed the Human Mesenchymal Stromal Cell Marker panel ab93758. Based on the information on the datasheet, I compiled the following information for you:
1. Positive selection (CD105, CD29, CD44, CD90)
2. Negative selection (CD45)
The stem cell panel ab93758 consists of 5 unconjugated primary antibodies. These antibodies can be used with the secondary antibodies against mouse or rabbit of your choice. You can therefore adapt the fluorochrome choice to the ability of your machine.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Hello! I have pasted the protocols below. Please let me know if you have any further questions!
Proteinase K Solution (Method 1) (20 ug/ml in TE Buffer, pH 8.0):

TE Buffer (50mM Tris Base, 1mM EDTA, 0.5% Triton X-100, pH 8.0):
Tris Base -------------------------------- 6.10 g
EDTA ------------------------------------- 0.37 g
Triton X-100 ---------------------------- 5 ml
Distilled water ------------------------- 1000 ml
Mix to dissolve. Adjust pH 8.0 using concentrated HCl (10N HCl). Store at room temperature.

Proteinase K Stock Solution (20x, 400 ug/ml or 12 units/ml):
Proteinase K (30 units/mg)----------- 0.008 g (8 mg)
TE Buffer, pH8.0 ---------------------- 10 ml
Glycerol --------------------------------- 10 ml
Add proteinase K to TE buffer until dissolved. Then add glycerol and mix well. Aliquot and store at –20 ºC for 2-3 years.

Working Solution (1x, 20 ug/ml or 0.6 units/ml):
Proteinase K Stock Solution (20x) ------ 1 ml
TE Buffer, pH8.0 ------------------------- 19 ml
Mix well. This solution is stable for 6 month at 4 ºC.

Proteinase K Solution (Method 2) (20 ug/ml in TE-CaCl2 Buffer, pH 8.0):

TE-CaCl2 Buffer (50mM Tris Base, 1mM EDTA, 5mM CaCl2, 0.5% Triton X-100, pH 8.0):
Tris Base -------------------------------- 6.10 g
EDTA ------------------------------------ 0.37 g
CaCl2 ------------------------------------ 0.56 g
Triton X-100 ---------------------------- 5 ml
Distilled water ------------------------- 1000 ml
Mix to dissolve. Adjust to pH 8.0 using concentrated HCl (10N HCl). Store this buffer at room temperature.

Proteinase K Stock Solution (20x, 400 ug/ml or 12 units/ml):
Proteinase K (30 units/mg) ----------- 0.008 g (8 mg)
TE-CaCl2 Buffer, pH8.0 --------------- 10 ml
Glycerol --------------------------------- 10 ml
Add proteinase K to TE-CaCl2 buffer until dissolved. Then add glycerol and mix well. Aliquot and store at –20 ºC for 2-3 years.

Working Solution (1x, 20 ug/ml or 0.6 units/ml):
Proteinase K Stock Solution (20x) ------ 1 ml
TE-CaCl2 Buffer, pH8.0 ------------------ 19 ml
Mix well. This solution is stable for 6 month at 4 ºC.

Procedure:
1.
Deparaffinize sections in 2 changes of xylene, 5 minutes each.
2.
Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each.
3.
Rinse in distilled water.
4.
Cover sections with Proteinase K working solution and incubate 10-20 minutes at 37 ºC in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user).
5.Allow sections to cool at room temperature for 10 minutes.
1. Rinse sections in PBS Tween 20 for 2x2 min.
2. Block sections with for 30 minutes.
3. Perform avidin/biotin blocking if necessary.
4. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 ºC.
5. Rinse sections with PBS Tween 20 for 2x2 min.
6. Block sections with peroxidase blocking solution for 10 minutes.
7. Rinse with PBS Tween 20 for 3x2 min.
8. Proceed to standard immunohistochemistry protocol.
Notes:
1. This method tends to cause tissue damage for under-fixed tissues. Select appropriate incubation time (5-20 minutes) and temperature (20 ºC to 60 ºC) for a specific application and do not over-digest tissues especially for the under-fixed tissues.
2. Method 2 contains CaCl2, where the Ca2+ can activate proteinase K enzyme by 20-25%, therefore increase enzyme activity.
Solutions and Reagents:

Pepsin Stock Solution (1% in 10mM HCl):
Pepsin ------------------------------------- 100 mg
10mM HCl (pH 2.0) ---------------------- 10 ml
Mix to dissolve. Store at -20 ºC

Pepsin Working Solution (0.5% in 5mM HCl):
Pepsin Stock Solution (0.5%) ------------ 1 ml
Distilled water ---------------------------- 1 ml
Mix well.

Procedure:
1.
Deparaffinize sections in 2 changes of xylene, 5 minutes each.
2.
Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each.
3.
Rinse in distilled water.
4.
Cover sections with pepsin working solution and incubate for 10-20 minutes at 37 °C in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user).
5.Allow sections to cool at room temperature for 10 minutes.
1. Rinse sections in PBS Tween 20 for 2x2 min.
2. Block sections with for 30 minutes.
3. Perform avidin/biotin blocking if necessary.
4. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
5. Rinse sections with PBS Tween 20 for 2x2 min.
6. Block sections with peroxidase blocking solution for 10 minutes.
7. Rinse with PBS Tween 20 for 3x2 min.
8. Proceed to standard immunohistochemistry protocol.
Note: This method tends to produce tissue damage so incubation time is import factor to consider when using this method. Select appropriate incubation time for a specific application.

Thank you for contacting us. I have placed information abou the trypsin solution and enzymatic antigen retrieval below.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Enzymatic retrieval, pipetting method
Materials and reagents
37°C incubator
Humidified chamber (either the incubator itself or a container with a wet paper towel)
Two slide rack containers of TBS with slide rack (See section G for TBS recipe.)
Enzymatic antigen retrieval solution (For trypsin, see below. For pepsin and proteinase K, see section G)
The following method uses trypsin. There are commercially available trypsin preparations optimized for IHC, such as Abcam's Trypsin enzymatic antigen retrieval solution (ab970), or it can be prepared as follows:
Trypsin Stock Solution (0.5% in distilled water)
Trypsin 50 mg
Distilled water 10 ml
Mix to dissolve. Store at -20°C.
Calcium Chloride Stock Solution (1%)
Calcium chloride 0.1 g
Distilled water 10 ml
Mix well and store at 4@°C.
Trypsin Working Solution (0.05%)
Trypsin stock solution (0.5%) 1 ml
Calcium chloride stock solution 1% 1 ml
Distilled Water 8 ml
Adjust pH to 7.8 with 1N NaOH. Store at 4°C for one month or -20°C for long term storage.
Method
1.Prepare the trypsin and pre-heat to 37°C. Carefully blot excess water from the around the tissue section and pipet the enzyme solution (generally 50 - 100 ul will suffice) onto the section. It may be necessary to spread the solution around the section with the pipet tip; be careful not to damage the tissue.
2.Place the slides in a humidified container and then into the 37°C incubator. Avoid placing the slides directly on the incubator shelves as there will be variations in temperature that could affect staining intensity. Ideally, the container holding the slides is pre-heated in the incubator.
3.After 10- 20 minutes (this will need to be optimized), remove the slides from the incubator and transfer to a rack in a container of tap water. Rinse by running tap water for 3 minutes.
4.Continue with immunohistochemical staining protocol.
Enzymatic retrieval, immersion method
Materials and reagents
37°C waterbath
Slide racks and slide rack containers
Enzymatic antigen retrieval solution (For trypsin, see pipetting method. For pepsin and proteinase K, see section G)
Method
1.Set water bath to the optimal temperature for the enzyme you are using. Add ultrapure water to two containers that can hold slide racks. Place the containers into the water bath to warm. (See note ii).
2.Deparaffinize and rehydrate sections as above. Place slides in one water container to warm (See note iii).
3.Prepare the enzymatic antigen retrieval buffer from the warm water in the other container, and then return the container to the water bath to allow the solution to re-heat (See note iv).
4.Transfer the warmed slides into the enzyme solution for 10 - 20 minutes (see note v) with intermittent gentle agitation, then remove the slides and place them in running tap water for 3 minutes to rinse off the enzyme.
5.Continue with immunohistochemical staining protocol.
Notes
i. Be sure to read the manufacturer’s literature for the enzyme you choose, as some enzymes require specific buffers and cofactors for activity.
ii. Use a sufficient volume of water or buffer to cover the slides.
iii. Placing cold slides into the enzyme solution will lower the temperature of the solution, reducing enzyme activity and leading to under-retrieval of the antigenic site.
iv. Prepare the enzymatic antigen retrieval solution as quickly as possible to avoid impairing the activity of the enzyme. Allow this solution to return to temperature before introducing the slides.
v. Ten to twenty minutes is only suggested as a starting point incubation time. Less than 10 minutes may leave the antigens under retrieved, leading to weak staining. More than 20 minutes may leave them over retrieved, leading to non-specific background staining and also increasing the chances of sections dissociating from the slides or damage to the morphology of the tissue. A control experiment is recommended beforehand, where slides of the same tissue section are incubated in the enzyme solution for 10, 15, 20, 25, and 30 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

Thank you very much for your calls today and for letting us know about the trouble with ab44967 and these other CD105 antibodies.

As we discussed, I'm sending a free of charge vial of ab11414 on the order ***, which should arrive tomorrow. Please keep me updated about the results with this new antibody, and let me know if there is anything else that we can do for you.

Have a nice day!

Thank you for your enquiry and interest in our products.

I can confirm that this panel contains 5 primary antibodies which recognize human mesenchymal stromal cells: ab6124, ab10559, ab23894, ab44967, ab52971. The datasheets can be found at these sites:

https://www.abcam.com/cd44-antibody-f10-44-2-ab6124.html

https://www.abcam.com/cd45-antibody-ab10559.html

https://www.abcam.com/cd90--thy1-antibody-af-9-ab23894.html

https://www.abcam.com/cd105-antibody-105c02-ab44967.html

https://www.abcam.com/integrin-beta-1-antibody-ep1041y-carboxyterminal-end-ab52971.html

There are some very informative and useful websites summarizing the different CD markers. You may wish to recommend to your customer for further consideration:

https://docs.abcam.com/pdf/immunology/cdantigen_poster.pdf

http://pathologyoutlines.com/cdmarkers.html

If you need any further assistance in the future, please do not hesitate to contact me.

Just wanted you to know I have found your order on our system. I will follow up with you shortly when have set up the replacement order.