Everest Biotech/CD34 Immunohistochemistry Kit 20 slides/3100070205/20 slides

CD34 is a type I transmembrane glycophosphoprotein which is highly expressed on hematopoietic progenitors, as well as on endothelial cells, brain, and testis. Staining for CD34 has been used to measure angiogenesis, which reportedly predicts tumor recurrence. CD34 is possibly an adhesion molecule with a putative role in early hematopoiesis by mediating the attachment of stem cells to the bone marrow extracellular matrix or directly to stromal cells. It could act as a scaffold for the attachment of lineage specific glycans, allowing stem cells to bind to lectins expressed by stromal cells or other marrow components. CD34 is thought to have a role in presenting carbohydrate ligands to selectins. The intracellular chain of the CD34 antigen is a site of phosphorylation by activated protein kinase C, suggesting a putative role in signal transduction.

Image shows immunohistochemical staining of paraffin‐embedded human Renal Cell Carcinoma xenograft tumor section stained with CD34 antibody using the Eton Bio’s CD34 IHC Kit (Cat No. 3100070105). CD34 (dark brown) displays a vasculature pattern which correlates its expression on endothelial cells (20X, counterstained with hematoxylin).

Protocol

The CD34 Immunohistostaining Kit has been designed for the staining of tissues that have been fixed (usually in neutral buffered formalin) and subsequently embedded in paraffin before sectioning. This protocol is recommended as a starting point and optimization by the individual end‐user may be required.

Note:

• Do not allow specimens to dry during the staining procedure. Specimen drying may cause increased non‐specific staining and background.

• Some tissue may need to bake to remove over‐covered paraffin prior to the procedure. If needed, bake at 55‐60°C for 30 minutes.

I. Deparaffinization and rehydration

Prior to staining, tissue sections must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section. Use positive control slide provided in the kit for quality control and trouble-shooting purpose.

1. Immerse slides in xylene and incubate for 5 minutes. Repeat twice with fresh xylene for another 5 minutes each.

2. Immerse slides in 100% ethanol for 5 minutes, and follow with immersion in 95%, 75% and 50% ethanol for 3 minutes each.

3. Rinse slides with distilled water for 5 minutes; keep in water until ready to perform antigen retrieval.

II. Heat induced antigen retrieval (HIAR)

Most formalin‐fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. Heat induced antigen retrieval can be performed using a steamer, pressure cooker, or a microwave oven. The

retrieval time written in this protocol is based on using a steamer. The heating time may need to be adjusted if you use a different device and method.

1. Fill plastic coplin jar/container with Antigen Retrieval Buffer (0.01M Citrate Buffer, pH6.0, not included in the kits).

Prepare Stock Solution:

0.1M Sodium Citrate 20.5mL

0.1M Citric Acid 4.5mL

Add distilled water to 250mL

2. Place the coplin jar/container in steamer with lid.

3. Turn on steamer and preheat to 90‐100°C. Carefully put slides into the coplin jar/container and steam for 40 min (95‐100°C).

4. Turn off the steamer, remove the coplin jar, place at room temperature and allow slides to cool for 20 min. Keep the jar covered all the time.

5. Rinse slide by incubation of slide in distilled water for 3 minutes. Repeat this step twice and begin staining procedure.

III. Staining procedure

Blocking of Endogenous Peroxidase

Note: Peroxidase Blocking is optional. If no non-specific staining is observed, skip these steps and go to step 3.

1. Tap off excess water. Draw a circle around the specimen on the slide with PAP pen (not included in the kit. Alternatively, folded Kimwipes could be used to briefly blot the water around the specimen. Repeat this blot step each time before add reagent on slide). Apply 200μl or more of Peroxidase Blocking Solution (not included in the kit) sufficient to cover specimen, and incubate for 5 minutes.

2. Rinse slide by incubation of slide in distilled water for 3 minutes. Repeat this step twice.

3. Rinse slide by incubation slide in PBS for 3 minutes.

Blocking of Non-specific binding

4. Tap off excess PBS. (If the Peroxidase Blocking step is skipped, draw a circle around the specimen on the slide with PAP pen or using the edge of folded Kimwipes to quickly blot the water around the specimen). Apply 200μl 1X Blocking Buffer immediately to cover specimen and incubate in a moist chamber for no more than 10 minutes

Note: 10X blocking buffer may form precipitates at 4°C. Completely dissolve the precipitates before making working solution

5. Rinse slide by incubation slide in PBS for 3 minutes.

Primary Antibody

6. Tap off excess PBS. Apply 200μl Equilibrium Buffer immediately to cover specimen and incubate in a moist chamber for 30 minutes

7. Tap off excess Equilibrium Buffer. Apply 200μl diluted anti‐mouse CD34 antibody (recommend 1:50 dilution in Antibody Diluent) to cover specimen immediately and incubate in a moist chamber overnight at 4°C.

8. Rinse slide by incubation in 0.5-2mL Wash Buffer for 3 minutes. Repeat this step twice with fresh buffer.

9. Rinse slide by incubation of slide in PBS for 3 minutes.

Secondary/HRP Conjugates

10. Tap off excess PBS. Apply 200μl Rat HRP Polymer immediately to cover specimen and incubate in a moist chamber for 60 minutes.

11. Rinse slide by incubation in 0.5-2mL Wash Buffer for 3 minutes. Repeat this step twice with fresh buffer.

12. Rinse slide by incubation in with PBS for 3 minutes.

DAB Chromogen

13. Tap off excess PBS. Apply enough DAB Substrate Solution to cover specimen immediatly. Check dark brown color development under microscope and incubate until desired stain intensity develops.

To make 1mL DAB Substrate Solution, mix the following reagents:

Distilled Water 860 μL

10X DAB substrate buffer 100 μL

0.3% Hydrogen Peroxide solution 15 μL

DAB Chromogen 25 μL

14. Rinse slide in tap water for 3 minutes.

Counterstaining

15. If desired, complete counterstain (See instruction for hematoxylin counterstaining). Rinse in tap water to clear.

Mounting

16. Immerse slides in 70%, 80%, 95% Ethanol for 2 minutes each, and 100% Ethanol for 10 minutes twice followed by Xylene for 5 minutes twice.

17. Dry and mount slides.

IV. Instruction for Hematoxylin counterstaining

1. Immerse slides in hematoxylin solution. Incubate for 30 seconds to 5 minutes, depending on the strength of hematoxylin used.

2. Rinse to clear with tap water and continue dehydration from Step 16.

Problems	Possible Causes	Solutions
Over staining	1. Too long incubation time of primary antibody, or too high temperature when doing staining 2. Too long incubation time of DAB substrate. 3. Slide dried during staining process	•Depending on tissue sections, the incubation time of primary antibody can be reduced to 2 hours; Check the room temperature range is at 20-25 0C when doing staining. •Reduce incubation time of DAB substrate •Avoid sections to dry during staining process.
Weak or no staining	1. Incomplete removal of paraffin 2. Tissues over‐fixation 3. Not efficient antigen retrieval 4. Reagents not used in proper order or omitted steps 5. Expired antibody or reagents	•Deparaffinize sections longer or change to fresh xylene; some tissue array may need to bake to remove over‐covered paraffin. •Increasing the concentration of primary antibody to 1:40; if this does not work, reduce duration of post‐fixation. •Adjust antigen retrieval time based on the setting for section fixation and retrieval device used. •Review notes and procedure used. •Check kit expiration dates and kit storage conditions
High background	1 Sections dried during staining process 2 Slide not rinsed thoroughly 3 Antigen over‐retrieval	•Do not allow sections to dry during staining process; use humid container during incubation with primary antibody. •Use fresh solution in buffer jars; rinse at least three times between steps. •Optimize antigen retrieval time if you used microwave or pressure cooker for retrieval.