Melisa, admitting I am not the specialist to critisize or comment any of the processing steps yo mentioned in your question (there will be other experts who will help with regard to antibodies, the IHC-technique and "no staing results", I guess ...), but:

the only thing I would like to  address you should think over is:  

"...so I will try liquid nitrogen next time). ".

It might be that you get some ( other and often severe ) problems in the handling as well as further "processing" = mounting in/with OCT and sectioning afterwards). I recommend to "cryofix" (as e.g. native, human muscle biopsies are frozen "state-of the art" ) with precooled Isopentane (= Isopentane in a separate vial will get slurry/ solid when cooled in LN2 and acts more rapidly and thoroughly than LN2 on its own...)  cf: e.g. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215994/      find section 4.: Muscle Freezing for Histological, Biochemical, and Molecular Studies

http://www.feinberg.northwestern.edu/research/docs/cores/mhpl/tissuefreezing.pdf

unfortunately (;-  only PPV(pay-Per-View):  https://www.jove.com/video/52793/do-s-don-ts-preparation-muscle-cryosections-for-histological;

or (this is not the end of possibilities): www.mdpi.com/2227-7382/4/4/32/pdf  , find in 2.3: Subjects and Tissue Preparation: Biopsies were mounted with tissue freezing medium, cryofixed in isopentane (􀀀=-(minus)160 =C) cooled

on liquid nitrogen and stored at 􀀀=-(minus)80 =°C. Serial cross-sections (10 = µm thick, 10 mm2) were performed using a cryostat (Microm, Francheville, France) at 􀀀= -(minus)20 =°C."

Best wishes and regards.

Hi Melisa,

1. Since you work with frozen sections you don't need antigen retrieval. I use it only in paraffin tissue sections where paraffin embedding could mask your antigen.

2. Once you cut your frozen sections in cryostat, leave the samples to dry at room temperature for 15 min and than store them at -80*C until staining. I wouldn't leave the frozen sections whole night at room temperature.

3. Is it really necessary blocking in 10%BSA? It could be enough 3% BSA, as well.

4. I haven't seen the secondary antibody incubation? I see you incubate with antibody mix 1 h, but you don't specify what are those antibodies. If they are primary antibody and the incubation is for 1 hour, where are the secondary antibody?

Best regards.

Hi Melisa,

Comparing your protocol of spleen extraction and immunochemistry with our own, I cannot see many differences. However, there are some steps that I think you could give a try and optimize in order to obtain better outcomes.

1) We fix using formalin-aqueous solution and saccharose but we freeze them using isopentane (as Wolfgang has said) at -35ºC during 30 seconds and then we store them at -80ºC until they are cutted. In our case we perform free-floating immunochemistry, so and additional formalin fixation (or acetone at -20ºC in the freezer, we prefer using formalin). 

Moreover, you could try to perform an O/N incubation of the primary antibodies to obtain the maximum staining. It is obvious but remember to use a secondary antibody if this is not a direct immunochemistry.

Hope to hearing from you soon,

J.

Hi everybody! 

Thanks for the replies and the suggestions!! 

Could I use isopentane with dry ice? Instead of LN2? 

The antibodies are conjugated already, that is why I did not use secondaries. But it would be a great idea to see if I can increase the signal... if the antigen retrieval is just for parafin, then my problem is in the magnitude of the signal I guess. 

Thanks again for your recommendations! 

Will tell you how eerything goes as soon as I test everything!

Hi Melisa,

regarding your request about freezing in isopentane "incubated" or pre-cooled by dry ice:

apologize if I first appear like a teacher with the following:

Physics behind freezing isopentane with LN2, until the very last point just PRIOR Isopentane becomes solid.

"Freezing point" of isopentane is  – (minus) 255.8°F = –(minus)159.9°C = 113.3°K. This is almost double value above the recristallisation T (= minus 80°C) where tissue specimens of histological size -  on "slow" freezing -  faces huge ice crystal artefacts   And just at - say- minus 150 or 156 °C the fluid isopentane (if not disturbed) transits to a hypercooled sludge/slurry which is NOT totally solid.

So, when transferring your specimen into that hyper-precooled slurry you use the really low temperature of say minus 150° C initial T - being transferred to the specimens surface - and then immediately to be changed most rapidly with a high cooling rate with decreasing temperature further down to -196°C, the temperature of LN2. The (proper sized = small) tissue specimen will be frozen optimally due to proper cooling capacity of the device AND optimal, rapid cooling rate (= Delta °C/t) of that hypercooled sludge/slurry (which - besides being hypercooled - exerts an "adhesive" property to the tissue - contrary to LN2 at -180°C!, - which evaporates for some second on a "hot" surface*) which literally is due to the so called "Leidenfrost's phenomenon"). (*) HOT because of the high temp-difference of over 200 °C with regard to body or skin surface temperature.

CAUTION (as it is recommended also for the handling with/of LN2):

Because of the awkward adhesive properties of the hypercooled slurry I recommend you to wear PPE (personal protection equipment), i. e.  minimally: EYE GOOGLES, and suited gloves for handling cold substances/matter/fluids AND NOT to get in skin contact with that hyper-precooled Isopentane sludge/slurry .

Please: if you can't read specific literature or have guided practical training on freezing specimens "state-of-the-art", try to get substantial knowledge from reading at least some suited articles about "Optimal freezing of biological specimens , seek the advice / suggestions from experienced colleagues (and read at least  https://en.wikipedia.org/wiki/Isopentane   - find:  ["Isopentane is commonly used in conjunction with liquid nitrogen to achieve a liquid bath temperature of −160 °C. " and   "Isopentane is used, in conjunction with dry ice or liquid nitrogen, to freeze tissues for cryosectioning in histology. [7]",   where Reference [7] links to: http://www.uab.edu/research/administration/offices/ARP/ComparativePathology/Pathology/Histopathology/TissueSubmission/Pages/Freezing-Tissues-for-Cryosectioning.aspx.  in the latter document find (cited here only for convenience of other readers. This also will answer your question if you can use Isopentane, precooled only by dry ice (USE PELLETS/SNOW instead of Dry Ice plates):

" Freezing methods

One simple method is to use dry ice (-70°C) in block form placed in a styrofoam container. Place the filled cryomold on the block to freeze it. This method has the advantages of simplicity and safety, but does not freeze the tissue as rapidly as immersion in a freezing medium.

The method we prefer uses dry ice in pellet form. Place a small stainless steel bowl (or Pyrex or polypropylene beaker) in the bottom of a styrofoam container and fill the space around the bowl with dry ice pellets. Place some pellets in the bowl and slowly add isopentane (2-methyl butane) or acetone [personal NB: this will cool down to approx. minus -109.3°F or -78.5°C] . Work in a fume hood, of course, as these are flammable. When the pellets stop bubbling vigorously, the "slurry" is ready. Once you've filled the mold and oriented the tissue, immerse it in the liquid to freeze it.

Isopentane also can be chilled in liquid nitrogen (-176C). With the liquid nitrogen in a styrofoam container or Dewar flask, use a tongs to lower a stainless steel, Pyrex, or polypropylene container of isopentane into the liquid nitrogen. The isopentane will start to become opaque as it nears freezing [personal NB: = formation of slurry]. Take the isopentane out of the liquid nitrogen and freeze the specimen as described above. Chill the isopentane again as necessary for subsequent tissues. This method has the advantage of very rapid freezing.

Storage

Frozen tissues can be stored in a -80 freezer. If the tissues were frozen in a flammable freezing medium, take care to allow it to evaporate before placing the blocks in the freezer.

Even if frozen in embedding medium, tissues must be protected from drying, which can ruin them. Tightly wrapped foil envelopes and screw-top plastic centrifuge tubes are commonly used, and it's a good idea to double wrap the specimens or place them in a container within a second container. Best, however, is to section and stain them as soon as possible.

See Cryotechniques for Light Microscopy © Woods and Ellis 2000 for more information. End of citation.

Also, last but not least, visit and read: https://en.wikipedia.org/wiki/Liquid_nitrogen, it is worth at least for basic, if not many specific information about properties too.

Best wishes, good luck, and have a beautiful weekend!