I had the same experience as you had, no it wasnt worth even trying, and I do see people do report those studies!!!

I have an alternative which should work very well though expensive: we just have to use cells that are expressing GFP or RFP or some other fluorescent protein, and then subjected to the 3D culture in matrigel. Now you have the luxury of monitoring the growth of 3D network over time with no pain of fixing, staining, etc. Alternatively, you can use cells dissociated from a tissue derived from a GFP mouse, which is also an expensive affair.

I had the same experience as you had, no it wasnt worth even trying, and I do see people do report those studies!!!

I have an alternative which should work very well though expensive: we just have to use cells that are expressing GFP or RFP or some other fluorescent protein, and then subjected to the 3D culture in matrigel. Now you have the luxury of monitoring the growth of 3D network over time with no pain of fixing, staining, etc. Alternatively, you can use cells dissociated from a tissue derived from a GFP mouse, which is also an expensive affair.

Thank you for the kind advice!

I have realized that not a few researchers have the similar problem in this kind of experiment.

I will try to establish YFP or GFP-stably expressing tumor cells with fibroblasts to perform F-IHC but also WB derived from each cellular population after cell sorting. However, I will stain molecule of interest in YFP or GFP-stably expressing tumor cells with primary antibody, so that I will try to prepare the frozen sections on slides instead of direct staining in order to improve the antibody permeability.  

If you use GFP or YFP-expressing cells for the assay, you should be able to image the network formation without any more processing, fixing, permeating/staining etc., by using a good fluorescence microscope, provided you are using appropriate dishes for the assay. Another advantage is that you can monitor the network formation, if you want, every day or whenever you want until you see the maximum response or until the end of your set protocol.

Good luck.

I suggest paraffin wax technique, in case you do not have access to GFP expressing cells or need to co-stain for other receptors.

After removing in-vivo matrigel plug, we fix it with 4% PFA, embed it in paraffin wax, then section and mount it on microscope slide. Each section is then rehydrated before immuno-staining. This works perfectly well.

The cryo-section cannot preserve the structure.

Please check this link for general information about this technique:

http://histology.leeds.ac.uk/what-is-histology/histological_sections.php

Labeling your cells with GFP or YFP before embedding in 3-D matrix will definitely work. Before you commit the time and effort into this strategy you might try "loading" your cells  with one if the Cell Tracker Fluorescent markers.  These are cytoplasmic non toxic dies that will persist for a certain period of time.  So it will depend what the duration of your experiment/study is if this approach is applicable.  Otherwise it will be as useful as GFP/YFP.  I would  suggest using Cell Tracker Red because it provides better contrast. The cell tracker dies are also useful if you intend to work with multicellular organoids  since you can prelabel each cell type with its own fluorescence..  Pre-labeling the cells takes only a few hours of incubation and is not medium dependent.. Good luck. 

Dan Dimitrijevich   

I also agree with the previous comments regarding GFP/RFP/YFP expressing cells or using cell tracker if your cells don't divide rapidly/your experiment does not go on for too many days. I've used both and it works quite well if you want to monitor cells over time.

However, if you need to section your 3D cultures to assess different markers you could either remove them from the matrigel (there are special products available for this) or you could embed the whole matrigel plug in 4% agarose (after fixation in PFA). This would keep your 3D cultures together, so you won't loose them when embedding in paraffin (see attached publication below). I hope you find a way that works, good luck ;-)

Article Advances in establishment and analysis of three-dimensional ...

Hi, 

yes Miriam advise will be good to try following the instractions on our publication mentioned above though keep in mind you will have to section your 3D culture and if for spheroids it is fine , I think when you have 3D invasion you would prefer to keep the complexity of the invasive network. In this case you will need to do a whole mount staining. I am currently doing that after fixing the invasive spheroids embedded into matrigel in 4% PFA, over night. Then you can stain for specific markers of your choice. You will need though to acquire images in z-stack on a confocal microscope. It sounds long procedure but the quality of the images is great. 

Hope this helps. 

Paraffin embedding of Matrigel and sectioning works well. If you have the possibility, whole mount staining followed by OPT gives the most beautiful little movies. 

 You can stain efficiently and clearly the 3-D cultured cells by using the same protocols used for 2D cultured cells. For fluorescent and/or visible probes, incubation times way have to be slighlty longer (compared to 2D) du ensure fair staining of cells inside the hydrogel but the hydrogel hasn’t any detrimental effect on probe/sample interactions.

It s really efficient with our Ha scaffold due to its transparency.

You might find this article by Mina Bissell to be helpful: Three-dimensional culture models of normal and malignant breast epithelial cells

http://www.nature.com/nmeth/journal/v4/n4/abs/nmeth1015.html

Hi

I'm interested in the handling of the matrigel plugs for processing to wax.

Is everybody fixing prior to embedding in agarose for processing or is anybody fixing once in the agarose. If so has this affected any markers you are using.

We did Matrigel in mice, formal fixed and paraffin embedded using standard hits protocols. A few were shrinking a bit, the rest was fine. For staining, if you have an antibody working on paraffin sections, it will work also using this protocol. 

Article The Wilms’ tumour suppressor Wt1 is a major regulator of tum...

If you have enough cells and are using organoids, you can remove the matrigel with corning cell recovery solution, and then do antibody staining in suspension (basically the same as how you would do it for FACs). After the nuceli staining step I resuspend in mounting anti-fade solution, and then pipette onto glass slide with coverslip as normal.  Its more time consuming, but you get much better signal

Hey everyone, I know this is an old thread but this might help:

If you don't want to embed and then section you can fix and stain directly but not with PFA. PFA will depolymerize (i.e. liquefy) Matrigel! So you aspirate all your precious cells during your washes. To prevent this you have to use gluteraldehyde, or a mix of PFA and gluteraldehyde. See Debnath et al publication with Brugge. They use a mix, which works for at least some immunofluorescence protocols. If you also grow the cells in a chamber that is suitable for imaging, then you can do all your fixation more or less as you would for 2D culture, and then image directly without the bother of embedding and sectioning.

Check this paper for a detailed protocol on immunostaining of intact organoids/spheres. Hope it helps.

https://www.ncbi.nlm.nih.gov/pubmed/30548444

Generation of Pancreatic Ductal Organoids and Whole-Mount Immunostaining of Intact Organoids