Quite frankly: I‘d try (at least- since P12 is relatively ‚young’ and tenuous) a lower PFA - concentration (e.g., 2%) …. but since you don‘t describe the whole collection and primary fixation protocol (mentioning precisely the steps of fixative making) it might be difficult to advise properly. ——— There was a time long long ago when [FA/Formalin/ PFA/formaldehyd] was classified to be a „dehydrating“ fixative… and for the tenious tissue fragments, 4% PFA (plain = nothing but hydrous FA made from PFA powder [how was it generated?] ? / buffered?? ) 30 min (RT?

, 4 degr. C?) may be way too a long fixation time

My two cents … [ unfortunately, answering delicate questions on RG using iPhone/RG App is quite unappealing due to restricted numbers of words ( only half of my post until —— above)

Hi Wolfgang,

Thank you very much for your kind answer. We dissection the eyes and directly incubate the retinas in PBS PFA 4% at room temperature for 30 minutes. This works perfectly for adult retinas and gives very good results in immunofluorescence. I think we will try to decrease the PFA concentration as you suggested and maybe incubate at 4 degrees instead of RT. Thank you again for your help!

Juliette

Dear @Juliette Bitard , now from my PC...first of all: apologies for my asking details you provided in your original question...Using the iPhone (RG-App) I was NOT able either to correct, or "copy" the text , in the end also to cancel my answer. So hopefully this time (using my RG-Account via PC) it will be possible to profit from well-functioning website settings.

Secondly: apologies for the lenghtiness of my (hopefully) more profound answer/post:

Thank you for replying that fast. Since - with regard to my 'studies' on RAT brain ('dissertation' / thesis in 1980; PN1, 6 12, PN 36, PN 120 - ....up to 140 days old)- I had to take into considerations age-related differences in fixation mechanism (immersion -, perfusion fixation,..) and so I had to read a lot of articles to get some insight... Just to throw in some 'inconsistencies': "PBS" isn't the same as plain "phosphate buffer" - and since there are a lot of 'PBS-formulations' out there in the wild - it is recommended always to define which one has been used in the admixture...(==> especially with regard to osmolarity / tonicity? = isotonicity? ).

On RG there are - for sure - some posts which perhaps are worth to be read...e.g. from October 2018: https://www.researchgate.net/post/How-to-avoid-artifacts-during-cell-fixationhttps://www.researchgate.net/post/How-to-avoid-artifacts-during-cell-fixation;

(NB: QUOTE from their paper, p.-190: "...It is recommended to make fixative for mammalian tissues with a total osmolarity of approximately of 500~700 mosmoles. At this total osmolarity, the effective osmolarity is most similar to that of living organisms. For reference, the total osmolarity for soft tissues such as embryos is 300, 400 mosmoles... "end of quote) (PDF) How to Get Well-Preserved Samples for Transmission Electron Microscopy. Available from: https://www.researchgate.net/publication/312180178_How_to_Get_Well-Preserved_Samples_for_Transmission_Electron_Microscopy [accessed Apr 08 2025].Or - more 'scientifically and specialized': 'How to Get Well-Preserved Samples for Transmission Electron Microscopy' by PARK et al_2016:Article How to Get Well-Preserved Samples for Transmission Electron Microscopy

Contrary to such measures you can find the following in a data sheet @ https://www.himedialabs.com/media/TD/TCL119.pdf: Paraformaldehyde Solution, 4%

4% Paraformaldehyde in Phosphate Buffered Saline

From the DIRECTIONS:

>>For Immunohistochemistry:

Add 1-4 mm thick specimen in 15-20 times volume of

4% PFA and incubate at the rate of 1 hr per 1 mm of

tissue.

>>For Immunocytochemistry:

Cover the monolayer of cells grown with a sufficient

volume of 4% PFA. Incubate for 15-20 mins at room

temperature or overnight at 4°C.

(Quite frankly: I would NOT follow these application "recommendations" blindly!)

From the "Quality Control"-Section one can learn that "Paraformaldehyde Solution, 4% = 4% Paraformaldehyde in Phosphate Buffered Saline TCL119":

pH: 6.50 -7.50

Osmolality in mOsm/Kg H2O: 1450.00 -1650.00,

which - IMHO - for delicate tissue like the mouse eyes you prepare - and the purpose these should serve - is way too high.

But also "hard fact" scienctific publications might give some insight to your problem: e.g.:

MARGO & LEE, Fixation of whole eyes: the role of the fixative osmolarity in the production of tissue artifact. 1995 Jun;233(6):366-70.Graefes Arch Clin Exp Ophthalmol. doi: 10.1007/BF00200486. [found at Natl. Libr. of Med. - = PubMed website: https:__//pubmed.ncbi.nlm.nih.gov/7672624/

Article Fixation of whole eyes: the role of fixative osmolarity in t...

(I don't know here, whether the RG-LINK harbours the article or not....).

At Springer's website (Article Fixation of whole eyes: the role of fixative osmolarity in t...

[NB: For not always to create ONLY the (aforementioned RG-Link automatically when pasting a certain publication-URL, I separate the following URL to the Journal's website by inserting 2 underscores "__" in between https: and //www. ... (so please copy and paste the following in your browser and before executing 'RETURN', delete the 2 underscores __...: https:__//link.springer.com/article/10.1007/BF00200486 ]

the article is offered as PPV (Pay-Per-view only) at € 39,95 net price (Austria). You could read at least the Summary/Abstract, as well as the References. Last, but not least, it might be interesting to find a suited Osmolarity of the fixative (either plain or with ions / buffer(s) added) calculating and/or using Osmolality/osmolarity 'calculators' (e.g.: https://www.rxkinetics.com/iv_osmolarity.html)

Now I won't exaggerate anybodys knowledge, and I won't comment more about types of fixatives like KARNOVSKY's 1/1 or KARNOVSKY's 1/2-Fixative (==> e.g.... https://www.emsdiasum.com/docstechnicaldatasheet15720?srsltid=AfmBOooNxCpNGeuaOMeKZuBF3Cyn5I5tOUhp18LKzDxfbefludQpyKO_

(for immunohistochemistry -IHC / ICC- naturally think -ing of use without the addition of glutaraldehyde...)

or other suited fixatives. I always have made the fixatives I had to use by myself, also taking measurements after the admixture was finished by using an osmometer to precisely evaluate the osmolarity as well as a pH-meter... so I knew always, what was in and if it was the correct formulation.....

Thank you for your patience and eventually reading this my pamphlet until the end... Best wishes, good luck and regards, Wolfgang H. MUSS, SALZBURG, Autriche.

Intricacies perfusio with cold fixative for five minute before isolating the retina has given us better results. Try it on a non experimental animal. Best.

Dear Juliette Bitard , again,

A small Text-correction needed for the former answer [003] :

as above: >>(NB: QUOTE from their paper, p.-190: "...It is recommended to make fixative for mammalian tissues with a total osmolarity of approximately of 500~700 mosmoles. At this total osmolarity, the effective osmolarity is most similar to that of living organisms. For reference, the total osmolarity for soft tissues such as embryos is 300, 400 mosmoles... "end of quote) (PDF) How to Get Well-Preserved Samples for Transmission Electron Microscopy. Available from: https://www.researchgate.net/publication/312180178_How_to_Get_Well-Preserved_Samples_for_Transmission_Electron_Microscopy [accessed Apr 08 2025]. Or - more 'scientifically and specialized': 'How to Get Well-Preserved Samples for Transmission Electron Microscopy' by PARK et al_2016:Article How to Get Well-Preserved Samples for Transmission Electron Microscopy>On RG there are - for sure - some posts which perhaps are worth to be read... e.g. from October 2018: https://www.researchgate.net/post/How-to-avoid-artifacts-during-cell-fixationhttps://www.researchgate.net/post/How-to-avoid-artifacts-during-cell-fixation ;

....Or - more 'scientifically and specialized': 'How to Get Well-Preserved Samples for Transmission Electron Microscopy' by PARK et al_2016:

Article How to Get Well-Preserved Samples for Transmission Electron Microscopy

(NB: QUOTE from their paper, p.-190: "...It is recommended to make fixative for mammalian tissues with a total osmolarity of approximately of 500~700 mosmoles. At this total osmolarity, the effective osmolarity is most similar to that of living organisms. For reference, the total osmolarity for soft tissues such as embryos is 300, 400 mosmoles... "end of quote) (PDF) How to Get Well-Preserved Samples for Transmission Electron Microscopy. Available from: https://www.researchgate.net/publication/312180178_How_to_Get_Well-Preserved_Samples_for_Transmission_Electron_Microscopy [accessed Apr 08 2025].