Methodology for Immunohistochemistry. Learning Objectives:

Transcription

1 Proteomics Methodology for Immunohistochemistry Methodology for Immunohistochemistry A staining process for identifying the proteins location in cells, tissues by using antigen-antibody property. Immuno means antibodies which are used to bind and histo means tissue sample to be detected. Learning Objectives: After interacting with this learning object, the learner will be able to: Perform cutting and mounting of the sections. Define rehydrating of the sections. Choose antigen retrieval process. Carry out immunohistochemical staining process. Infer the sections under microscope. Assess the troubleshooting steps involved in the experiments. Note: The current IDD exists in two modes- interactive and automatic. Students taking lab course should select interactive (set as default), while the automatic mode may be selected for general users.

2 Fixing the Tissues Fixing the tissue is an important step and depending on the tissue size the fixation time varies anywhere between 18 to 24hours. The fixation process helps to fix the cells using a fixative such as neutral buffer formalin or paraformaldehyde solution.

3 Fixing the Tissues The fixed tissue must be embedded in paraffin wax. The tissue blocks with paraffin wax can be used for tissue embedding. This tissue block provides support with the paraffin holding the tissue at the time of sectioning.

4 Cutting into Sections The embedded tissue block must be placed in the microtome. The microtome helps in producing tissue of the required thickness usually defined by the user. As the cuts are produced, user can take out the sections with the help of the forceps. In the initial sections, fine slices of only wax can be seen but as the sections increase tissue slices embedded in paraffin are visible.

5 Cutting into Sections Once the sections are produced from microtome, they must be transferred to water bath, so that the paraffin wax gets melted leaving behind the tissue sections. Now pick the section exactly in the middle of the slide. Tissue sections must be mounted on positive charged slides.

6 Rehydrating the Sections The ethanol and xylene solution must be prepared for rehydrating the sections. Ethanol must be prepared in decreasing concentrations.

7 Rehydrating the Sections Xylene solution helps to remove the paraffin wax and alcohol treatment rehydrate the tissue sections. This step enhances the binding property of the cells making access to the interior easy.

8 Antigen Retrieval Prepare 1nM tris-edta buffer, it is used in antigen retrieval step. The ph of the buffer can be set to 9.0. These solutions can be prepared well in advance or can be prepared fresh during Xylene and ethanol treatment.

9 Antigen Retrieval Use sufficient volume of buffer in order to cover the slide. Keep a watch on the slides while boiling and avoid the slides from drying out. Less time may leave the antigens under-retrieved and more time may leave them over-retrieved. A series of experiments can help in better deciding the time for retrieving the antigen following which the user can proceed to immunostaining.

10 Staining of Sections Use sufficient volume of TBS buffer in order to cover the slide. The user can now proceed to immunostaining step.

11 Staining of Sections The concentration of working stock depends on the experiment. The working solution must be prepared in TBS depending on the concentration required for staining. The antibodies are very costly, so proper dilution for the working stock must be prepared by defining the experiment requirement. Store the working and the stock solution at -80 C.

12 Staining of Sections Each section must be treated with BSA to prevent nonspecific binding, later add primary antibody and place it for incubation overnight at 40 C. Incubation must be done in humidifier to avoid drying of sections.

13 Staining of Sections Mark the section boundary on the other side of the slide with a marker. Addition of the antibody must be sufficient enough to cover the complete section and must be within the marked boundary of the section. During primary antibody treatment, wash the slides with TBS to remove unbound primary antibodies. After primary antibody treatment, excess antibody solution must be cleaned out from the slide. Now add secondary antibody and incubate for 1hour inside a humidified chamber.

14 Staining of Sections The staining process depends on the user requirement, tissue used and the setup of microscope. Commonly used counterstains are hematoxylin (blue), nuclear fast red or methyl green. Once staining is over, the slide must go through dehydration step before viewing under microscope.

15 Dehydrating the Sections The dehydrating step is carried out to remove excess stain, if any, present in the slide. After the ethanol treatment, the slide with the section is placed in distilled water before proceeding to next step.

16 Microscopic Viewing The mounting solution like organic mounting media has better refractive index than aqueous mounting media. Now the slide is ready for viewing under microscope.

17 Microscopic Viewing For better image, fine adjust the microscope. Once confirmed, save the final image for use in publications and research articles.

18

19

20

21

22

23

24