Dear Vinesh,

There are several ways to prepare slides for hiistological analysis. I notice that you want to detect physiological changes, so you will need to embbed your samples in resin ou parafin.

I will give you a standard method for light microscopy.

First of all, you will need to colected the samples and fixate as soon as possible. You can fixate in several reagents. I'll give some examples: you can use FAA (Formaldeid, acetic acid and Alcohol 70% or 50%, or youn can use Karnovsky solution ( Karnovsky, M.J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J of Cell Biol 27, 137-138. )

During the fixation the samples needs to be submitted to a vacuum pump to remove the air inside the samples. I use 15 minutes for 3-4 times during the day.

After that you made the dehydration in an ethanol series (10, 30, 50, 70, 90, and 100%). - 10- 20 minutes in each ethanol grade.

When you finish the dehydration you need to make a transition of ethanol to resin (3:1, 1:1, 1:3,) the time for each step is variable. Depends the size of your sample. you can write to me indicating what plant tissue do you want to analyse. What is the size? I use a plastic resin (Leica Historesin®, Heraeus Kulzer, Hanau, Germany). But there is other companies that produce methacrylate resins.

But if you will use paraffin, you need to make a transition of ethanol to xilol (3:1, 1:1, 1:3) (one hour per step) and than other transion for xilol to paraffin (3:1, 1:1, 1:3). When you use paraffin you need to have one Oven at 60 C. Because paraffin is solid at room temperature. I send you a good protocol about paraffin in attached file.

The polimerization of resins happens at room temperature.

For cut your samples to do the slides you need a microtome.

Please let me know if you understand each step. If you have some questions feel free to contact me.

Good Luck!

Which kind of slides you want to prepare? What kind of structure you want to detect? This website may provide you almost all information you need.

http://dps.plants.ox.ac.uk/langdalelab/protocols.html

Sir, I want to detect the physiological changes in plant tissue.

What kind of physiological change do you want to detect? Expression level of proteins? Subcellular distribution of molecules? Or morphological change of cells?

Dear Vinesh,

There are several ways to prepare slides for hiistological analysis. I notice that you want to detect physiological changes, so you will need to embbed your samples in resin ou parafin.

I will give you a standard method for light microscopy.

First of all, you will need to colected the samples and fixate as soon as possible. You can fixate in several reagents. I'll give some examples: you can use FAA (Formaldeid, acetic acid and Alcohol 70% or 50%, or youn can use Karnovsky solution ( Karnovsky, M.J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J of Cell Biol 27, 137-138. )

During the fixation the samples needs to be submitted to a vacuum pump to remove the air inside the samples. I use 15 minutes for 3-4 times during the day.

After that you made the dehydration in an ethanol series (10, 30, 50, 70, 90, and 100%). - 10- 20 minutes in each ethanol grade.

When you finish the dehydration you need to make a transition of ethanol to resin (3:1, 1:1, 1:3,) the time for each step is variable. Depends the size of your sample. you can write to me indicating what plant tissue do you want to analyse. What is the size? I use a plastic resin (Leica Historesin®, Heraeus Kulzer, Hanau, Germany). But there is other companies that produce methacrylate resins.

But if you will use paraffin, you need to make a transition of ethanol to xilol (3:1, 1:1, 1:3) (one hour per step) and than other transion for xilol to paraffin (3:1, 1:1, 1:3). When you use paraffin you need to have one Oven at 60 C. Because paraffin is solid at room temperature. I send you a good protocol about paraffin in attached file.

The polimerization of resins happens at room temperature.

For cut your samples to do the slides you need a microtome.

Please let me know if you understand each step. If you have some questions feel free to contact me.

Good Luck!

Good day, I will be glad if you can send a good protocol to me about Plant histology, preparing permanent slides of plants samples

thank you