It is necessary to adjust right osmolality. If you send me the accurate composition of your fixative, I will compute the osmolality and I suppose the modification of the fixative.

Pavel

Dear Sir,

thank You for answering my question. My fixative compounds are:

100 ml 20% formaldehyde

2 ml 25% glutaraldehyde

250 ml 0,2M sodium cacodylate buffer

500 ml dH20

Is it enough or You need more specific information?I'll be very, very thankful for Your help!

With kindest regards

Karolina

Dear Karolina,

in the fixatives it is very important to calculate the effective osmolality, which cannot be measured by the osmometer. This is due to molecule size of the fixative components. Components such as formaldehyde have the molecule smaller than the water molecule and therefore they have no osmotic effect on the cells. Using osmometer we obtain the value non respecting this biological parameter. I calculated the effective osmolality of your fixative and it was at about 120 miliosmol/kg. We need for the successful effect of the fixation the value of 280 to 300 miliosmol/kg.

Therefore I suppose this composition:

100 ml 20% formaldehyde

2 ml 25% glutaraldehyde

340 ml 0,2M sodium cacodylate buffer

and distilled water complete up to 500 ml of total volume. I accent, complete up to, do not add 500 ml!

I hope it will be useful for you.

Pavel

Dear Sir,

of course, I should write 'completed up to 500ml with dH20'- my stupid mistake :)

Thank You very much for help. I let You know how this improvement works.

Karolina

Dear Sarah,

Thank You for Your answer. Could You please send me Your fixation procedure? It will be very helpful for me.

Regards,

Karolina

Dear Karolina, (& cherished fore-posters),

not knowing if you are going to start or already have done your fixation meanwhile:

at least 4 points to be elucidated - or taken into consideration - additionally:

i) you intend immersion fixation of (whole/lamellar sectioned) kidney(s) of

ii) human, animal (if latter, which one?)

iii) perfusion fixation of whole kidney by retrograde perfusion might yield best results.

iv) immunogold-labelling of uncomplicated epitope or something unusual?

Please remember that one of the "best fixatives" for Immuno-labeling studies that has been reported to be McLEAN&NAKANE's (1974)

PLP-fixative (= 1% (P)FA + 0.1M lysine, diluted in/with 0.1 M sodium phosphate buffer, pH 7.4 (resulting phosphate concentration is0.05 M).

To that fixative 0.1 % GA can be added for TEM-IEM-studies.

(Recipe - also for making fresh PFA = formaldehyde made from paraformaldehyde powder = absolute monomeric formaldehyde without traces of the usually contained stabilizer 10% methanol - could be provided)

Also one should bear in mind that kidney tissue could / should be fixed in a fixative whose tonicity (i.e. m-osmolality) is a bit higher than blood, resulting in a value of around 400 mosmol instead of 280 to 300 miliosmol/kg as usually for human tissues / isotonicity of hum. blood samples (isotonicity for kidney: approx. 0.23 M NaCl= 417-440 mosmol). Such an approach should be considered depending on the renal compartment you would like to study after tissue processing. Furthermore it has been reported that - depending on the age of your animal (if you study animal kidney) - the concentration of fixative might be a variable in the fixing system (e.g. Maunsbach 1966, Larson 1975). As Pavel already pointed out, the correct tonicity of the fixative (= fixing aldehyde (to a minor portion) + vehicle= buffer solution) is crucial, but swelling and / or shrinking issues upcoming also during deyhdration. So this might be also a point worth of discussion

best wishes and regards,

Wolfgang