Welcome to the Abcam blog

What you will learn

Our ICC webinar provides tips for:

• Cell culture and sample preparation
• ICC general staining procedure (Fixation and permeabilization, Antibody staining)
• Recommended controls
• Fluorescence
• Troubleshooting
• Antibody selection

Watch our immunocytochemistry webinar

Immunocytochemistry explained

Immunocytochemistry is a method for demonstrating the presence of protein within individual cells in cell culture. This is a useful application for many research areas which can be used to assess the specific localisation of proteins within the cell.

Our resident ChIP expert, Dr. Karen Halls, presented a webinar on an ‘Introduction to ChIP, Principles and Troubleshooting’.

Read the most common ChIP questions, and their answers of course.

• Does formaldehyde fixation need optimization?
• What type of ChIP is right for me?
• Is MNase digestion possible in X-ChIP?
• How long are your sonication cycles?
• Guidance on how to find promoter sequences for histone ChIP
• What are the best positive control genes and regions?
• Do you have any resources for identifiying sequence for ChIP primer design?
• Why is the chromatin fragment ideal size between 150 - 1000bp?
• How is chromatin or DNA value assessed?
• How do you prepare tissue for ChIP?
• Are there any recommendations when using a probe sonicator for the sonication step?
• If I use native ChIP, do I still need sonication?

 

The time of fixation with formaldehyde needs optimization by the researcher. Is that true because 2-30 min is a long time?

Answer: Generally this doesn’t need to be optimised, 10 minutes should be sufficient for cell culture or slightly longer for tissue samples.

 

If I am planning to study epigenetic modification especially for detection histone modification, what is the most suitable technique, X-chip or native one?

Answer: X-ChIP or N-ChIP would work well. We have more experience of X-ChIP so would probably recommend this technique. This tends to be a more straightforward method to get right.

 

Is MNase digestion possible in X-ChIP?

Answer: Yes, this is possible but it will not be as effective as sonication due to the protein/protein cross-linking. Some areas will not digest very efficiently. If you would like to perform enzymatic digestion, we would suggest cross-linking for reduced time and including an additional short sonication step to ensure DNA is fragmented sufficiently.

 

In your example of sonication optimization, how long are your sonication cycles? I presume you do not sonicate constantly for 20 seconds?

Answer: It was a 1 minute cycle with 30 seconds sonication, 30 seconds without, for 20 minutes.

 

Can you provide guidance on how to find promoter sequences for histone ChIP, and how to design primers that are specific to promoters that have modified histones bound to them?

We are doing ChIPs for histone modifications in our genes of interest. How do we identify good positive control genes and regions for designing our test primers, when histone marks are highly variable by cell type, and by publications?

Is there a website besides the University of California, Santa Cruz genome browser which is a reliable resource for identifying sequence for ChIP primer design?

Answer to the last three questions: As far as we are aware there is no database which shows all histone modifications across all genomic locations (this will vary by cell type and cell line). The following two links might help:

» Human Histone Modification Database (HHMD)

» ChromatinDB: A database of genome-wide histone modification patterns for Saccharomyces cerevisiae

For positive control genes, we would suggest trying some of the standard histone marks for gene activation and silencing and check the literature. There are websites and software that will assist with primer design. There will be software provided with real-time PCR machines. We have used Biotools primer tool 3 in the past, but others may be available.

 

Why is the chromatin fragment ideal size between 150 - 1000bp?

Answer: N-ChIP will be ~150 bp, X-ChIP will be ~500 bp. It must be larger for X-ChIP to ensure that intra nucleosomal DNA is not fragmented and nucleosomes lost

 

On slide 31 'Quantity of chromatin' is it indeed chromatin or DNA? How is this value assessed?

Answer: 25 ug of chromatin, this can be measured by Bradford assay. Use ~1x106 cells per ChIP sample

 

What is the procedure for preparing tissue from mouse brain? For example, do you perfuse the mouse with 1.5% PFA and then micro dissect the region of interest? Is there a minimal volume of tissue needed for detection?

Answer: We recommend to check out our ChIP tissue protocol for more information.

 

Are there any recommendations when using a probe sonicator for the sonication step? I feel like I'm not getting very consistency in my sonications

Answer: It is very difficult to obtain consistency with a sonicating probe. We would recommend to sonicate for very short bursts and rest the sample on ice. Foaming is the major problem when using a probe.

We would recommend a sonicating waterbath, as it provides reproducible results and minimzes sample foaming.

 

If I use native ChIP, do I still need sonication?

Answer: No it is not necessary to sonicate. Micrococcal nuclease digestion will be sufficient.

 

Q. Tell us a little bit about your backgrounds. What research are your labs currently working on?

Both of us are stem cell biologists working at the MRC Centre for Regenerative Medicine in Edinburgh.

Clare Blackburn:

I work on the generation and regeneration of the thymus, with a particular focus on fetal and adult thymic epithelial progenitor/stem cells. The overarching aim of my work is to develop new cell replacement or regenerative strategies for boosting thymus function in patients and in the elderly.

Val Wilson:

I work on the emergence, maintenance and disappearance of a newly defined stem cell type - the neuromesodermal stem cell - that exists only in the embryo and generates the spinal cord and musculoskeleton.

Q. What were your motivations to organize a conference on the Origins of Tissue Stem Cells?

The meeting is very timely due to recent advances in multiple systems, and therefore we wanted to bring together scientists working in these areas in order to promote discussion and catalyse progress.

Q. We have a line up of speakers and topics. What do think makes this meeting stand apart from other related meetings and who do you think would be most interested in this meeting?

This meeting covers an important area that is often neglected in larger meetings on stem cell research. Understanding the mechanisms leading to stem cell generation in vivo are fundamental to understanding the stem cell niche, and therefore maintenance and manipulation of stem cells in vivo and in vitro, and is particularly relevant for stem cell and developmental biologists. It has a great line-up ranging from some of the top established names in the field to the emerging stars of the future.

Q. You both work and live in Edinburgh. What can you say are the best things/highlights of Edinburgh?

Edinburgh is one of Europe's great cities, full of history and culture. It also offers fantastic restaurants and is home to the Scottish Malt Whisky Society. Come and visit before you need a passport!

Q. What sparked your passion for science?

It sounds a bit clichéd but it was an inspirational woman science teacher. She put me forward as a participant in a Youth Science fortnight in the UK. This was based in London, involved a series of talks given by academics, visits to drug companies as well as entertainments and a free travel card. I was 16 and had a great time. I met some really nice people with similar interests and enthusiasms.

Q. What were your motivations to organize a conference on Immune Tolerance and Autoimmune Disease?

I have organized conferences on this topic before but this was the first opportunity to organize a short conference in Cambridge. It seemed like a good idea.

Q. Where do you see things going? What do you think will be the next big breakthrough in this field?

I think that imaging techniques will continue to develop through interactions between physicists, engineers and biologists. This will further enable the tracking and study of inflammation in vivo at higher resolutions.

Q. How did you first connect with Abcam and come to chair this conference?

I had been in conversations with Kieran Matharu from Abcam and he mentioned that there might be a possibility of organizing a conference.

Q. What makes this meeting stand apart from other related meetings?

The format of interspersing short talks from participants between the invited talks is not so common. It is a smaller meeting and therefore has more of a workshop feel which would enable the participants to talk more readily with invited speakers.

Q. Who do you think would be most interested in this meeting?

I hope that it will interest a range of people including younger and more established scientists and clinicians. I also hope that it will attract interest from individuals working in biomedical research companies.

Q: What exciting research projects have been occurring in your lab?

We have been studying how organ size control mechanisms can impinge upon stem cell activity and differentiation. We are very excited about this line of research because the biology of organ and tissue size control are very poorly understood, so our findings have the potential to uncover novel paradigms and mechanisms that orchestrate stem cell behavior . This, we hope, will provide important insight for the manipulation of stem cells in the context of regenerative therapies.

Q: How did you first connect with Abcam and come to chair this conference?

Through my co-chair Laurie Boyer. She has been the chair of outstanding Abcam conferences in the past, and we decided that it would be a great time to have a meeting focused on stem cell biology and topics at the interface between basic science and clinical application.

Q: What were your motivations to organize a conference on Stem Cells to Tissues?

The field of stem cell research has moved at a frenetic pace over the past decade. While in general clinical application of basic stem cell research has not fully materialized, we felt that the scientific community is at a crucial juncture where basic insights are allowing some of these challenges to be overcome. Thus, we felt that this was a good time to get the leaders of the field together and highlight the latest advancements that might drive the field and potential clinical utility for years to come.

Q: What makes this meeting different from other stem cell meetings?

For a one-day meeting, the quality of the speakers is unparalleled. Additionally, though all the speakers have the common overriding interest of understanding the biology of regeneration, given their different backgrounds, the multiple models, perspectives and approaches presented make this a singular meeting.

Q: What new and exciting developments may we hear more of at the meeting?

I think we have an incredible line of speakers. We might hear the latest developments on the clinical application of epithelial stem cells for the treatment of disease, the use of novel mRNA delivery technology to treat heart disease, and the most recent insights into regenerative processes in model organisms.

Q: We have a great line up of speakers for the conference. Is there a particular topic or speaker you are looking forward to hearing?

I am particularly looking to hear from Doug Melton. He has really been a pioneer on the study of endodermal stem cells and the development of new approaches to understand pancreatic regeneration. I am also looking forward to hear from our keynote speaker, Hans Clevers. His work really represents what this conference is all about, pioneering basic science that can be translated into patient therapies.

Q: Who do you think would be most interested in this meeting?

Everyone with an interest in stem cell biology and regenerative medicine.

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In addition, this meeting is hosting a poster prize worth up to $1000. Please visit the Stem Cells to Tissues event page for complete rules and conditions.

Western blot (WB), also called immunoblot, uses specific antibodies to analyze one protein in a sample containing multiple proteins. Proteins are separated from each other by size using gel electrophoresis, then transferred to a membrane and detected with a specific antibody. This technique is widely used in biological research, as it provides information about the molecular weight of a protein as well as relative differences in expression between samples.