Hello, you may try CAF ( Calcium Acetate Formaldehyde) Besides formalin.

Hi Michal,

You my try agarose embedding of the lobes after postfixation and before cutting the slice. 5% low melting agarose works well for most of our tissues, but for lungs it might be a bit too dense so check also 4-3% one. Be careful that the poured mixture is not too hot. We usually pour the blocks using agarose at around 40 degrees (checked on the inside of your forearm- just like baby milk :) ). The agarose can be dehydrated and embedded in paraffin with the tissue inside and does not affect sectioning.

Also you my try to cut the samples not with scalpel blade but with fresh, disposable microtome knife. It helps for some people

Let me know if it helps. Also If you have other questions or want to know any details just PM, I am happy to help.

Good Luck :)

Thanks

Actually I do use preembeding in gelatin-agar, but later on, after slicing, to create a gel-block that is easy to handle during processing and paraffin embedding.

But it is a good idea to first dip lobes in more concentrated gel, to create a thin shell, and then slice them. I will go for it and try.

I have try the microtome knives – it may be very subjective, but I had an impression that such knives (at least the one I used, but freshly taken from the package) were even blunter than scalpel blades.

The benefit of microtome blade in our hand is that it is easier to get a seesaw motion with even distribution of force resulting in less of pressing downwards. But probably it is a matter of technique and personnal preferences.

And a question from me: does agar-gelatin not interfere with IHC? We got a nasty brownish background around our tissue once we tried it.

Hi,

there may be few factors influencing the reactivity of agar-gelatin in IHC:

- antibody – I can imagine that IHC for collagen may give a lot of background, since there is collagen in gelatin. I haven’t use such Ab, but with my Ab the gel gives very weak background and in prolonged DAB reaction (it may be also result of autoclaving described below)

- preparation of gelling solution – I have tested few conditions, concentrations and components of such solutions, and tested them in stainings I used in lab (trichromes, picrosiurius red, PAS, HE...) and the best result (with minimal background) was when used 2,2% gelatin and 2% agarose in PBS, which I autoclaved for 1h to “unfold” (?) collagen – if only microwaved, the collagen stainings were very intense. My solution remains liquid at 42-43oC

- fixation – I’m not fixing gel after preembedding, and wash tissue thoughtfully before preembeding

Still, sometimes there is some remains of “dirt” (which I believe is fixed gel, from remains on NBF in spongy structure of lungs), especially in alveoli, so right now I am trying to set the method to remove the gel from the section on slide before staining.

Thanks. Have not thought about autoclaving.

For pre-embedding we are currently using pure low melting agarose in the concentrations of 3-5% just microwaved. It is more expensive than agar-gelatin and is in my experience less flexible and tissue-like, but it removes itself in the dewaxing process and generally serves its purpose. But we will experiment a bit with the protocol you provided. It's always good to have more options available.

Btw, do you fix the gel-block after preembedding? Or do you wash tissue thoughtfully before preembeding to remove fixative? I don't know how agarose behaves in fixatives...

Our standard protocol is 16-18h fixation in comercial 10% NBF(HT501128 from SIGMA) followed by PBS wash. If tissues are stored after fixation they are kept in 1%NBF in PBS at 4 degrees, and briefly rinsed before embedding. The agarose we use is also from SIGMA (A9539). No postfix after embedding. Also we use automatic Microwave Hybrid Tissue Processor (Logos from Milestone). Don't think we ever used agarose blocks for manual dehydration and paraffin embedding.

Thanks, as you wrote - it is always good to have more information and experience :)

I fix the lungs sample in 10% NBF for 24 hours. Before cutting each section, I humidify the surface of the block for 1-2 seconds. Without humidifying, I also have artifacts. It's for each cutting.

I have an additional question : should the mouse be perfused with PBS first ?

I want to have feedback on experience to know, if it's better to get a washed lung for IHC quality while we loose some info, such as capillary easy identification or other stuff, or to not washed with the risk of having a "bloody" lung that may affect IHC ?

tks for your help