Dear Kristina,

If you have option to divide tissue into 3 categories, that will be good:

*For IHC, fix the tissue in cold 4% PFA (freshly made or freshly diluted from concentrated solution like from 16% which comes from EM sciences in glass capsules) and than pass thru 15% and 30% sucrose and than place the tissue in cryomold with OCT and freeze them on dry ice and store them at -70C or in LN2.

**For Gene expression: place the tissue in sterile cryo vial and throw in LN2 as soon as possible. Since it is is human tissue, taking tissue from patient takes time and during this time, gene expression can change a lot.

**Alternatively, place the tissue in sterile tube containing RNAlater (read RNAlater instructions for further storage), at RT for one to 2 days or 4C for a week, or freezing for longer time depending how long you want to store, all options available (this option is better)

***For Eliza,: you should freeze in aliquotes containing lysis buffer and protease inhibitors and storage at -80C. You can read this site: which is informative:

https://raybiotech.com/learning-center/preparing-cell-or-tissue-lysates-for-elisa-kits/

Dear you can keep the samples in aliquots, freeze and store other in liquid nitrogen.

To answre why do I have to block?

Free surfaces have to be blocked in immunoassays. This is true for many methods such

as ELISA, protein arrays, immuno-PCR, Western blotting or immunohistochemistry.

The surfaces of ELISA plates or Western blotting membranes are optimized for

efficient attachment of proteins and antibodies. This enables you for optimal assays with a

high density of capture antibodies or capture molecules on the surfaces.

As a consequence of this optimization of the surfaces, many other proteins or

peptides can attach on the surface. This attachment is reversible. Without blocking

of surfaces, assay antibodies, tracers, analytes or other components of the sample

would bind to the surface. This can lead to false results or high background signals.

Direct attachment of detection antibodies to the ELISA well leads to a signal even in

the absence of analyte. This has to be prevented. Blocking is necessary to saturate

free binding capacities of the respective surfaces. This saturation you can achieve it by

covering the surface with a dense layer of molecules without gaps in this layer.

After coating of the ELISA plate with the capture antibody or antigen, the free spaces

of the surface have to be blocked. A blocking solution contains a blocking agent in

high concentration. Incubation is done in the microwell. This leads to an adsorption of

the blocking agent to the free binding sites on the surface. Ideally a completely dense

and gap-free layer is generated. Incubation time depends on the blocking agent and

can be extremely short (in case of very good blockers). Generally it’s recommended

to use longer incubation times over night and to choose an appropriate operational procedure.

After blocking you can proceed with the assay. In addition to a blocking step a

subsequent stabilisation step with The Liquid Plate Sealer® is recommended. This can

improve binding capacity of capture antibodies to the analyte, and can improve

the results of some assays in terms of sensitivity and reproducibility. Additionally

it’s possible to store coated ELISA plates for longer periods before using them. This

stabilizer is incubated in the well of the ELISA plate as with the blocking agent. Liquid

Plate Sealer® covers the capture antibodies or coated antigens with a protective and

easily soluble layer.

This layer has two effects. First, the layer can directly improve the structural

conformation of many capture antibodies or antigens. The improvement in

conformation occurs during incubation and following the drying process of the ELISA

plate. This is demonstrated by a higher binding capacity of the capture antibodies

compared to untreated capture antibodies. This effect can be measured directly after

drying of the plate, when the effect is relevant to the coated capture molecules. The

second effect is a long-term stabilization of the coated biomolecules. ELISA plates

can be stored for long periods at 4°C after stabilization. In most cases operational

procedures in the lab can be facilitated by coating and storing many plates in stock.

Stabilizing can also be used to reduce coefficients of variations of ELISAs. The positive

effects of Liquid Plate Sealer® strongly depend on the biomaterials which are used

(antibodies and antigens). Therefore the positive effects cannot be quantified

generally but only based on the respective assay.

Regards