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IHC Select Detection Systems

Millipore's Premium IHC Select® Detection Systems offer the highest quality staining of formalin-fixed, paraffin-embedded tissues. Designed to work with any IHC validated antibody this kit contains all the necessary reagents for your IHC studies. Standardized protocols and prediluted reagents result in reproducible high quality staining in less than 1 hour.

Detection can be performed using either Horseradish Peroxidase/DAB or Alkaline Phosphatase/ Fast Red. IHC Select® Reagents may be purchased as complete kits or as individual components.

Rapid Results

  • Superior quality staining in less than 1 hour

Optimized Reagents

  • High Signal-to-Noise Ratio
  • Consistent results

Easy-to-use

  • Color-coded reagents
  • Simple protocols

Flexible

  • Ideal for manual or automated systems

IHC Select®

Complete Detection Kits include the following:

  • 20 X Rinse Buffer
  • Blocking Reagent
  • Biotinylated Secondary Antibodies
  • Streptavidin-Enzyme Conjugate
  • Chromogen (A and B components)
  • Hematoxylin

IHC Select® Manual Staining System

The purpose of the Chemicon IHC Select® Manual Staining System is to allow the simultaneous IHC staining of 2 to 40 microscope slides. The microscope slides are held in one slide holder and moved together throughout the IHC procedure. This minimizes technician time, and maximizes efficiency and reproducibility. Because this system makes IHC easy, more IHC experiments can be performed in less time and with less technician stress.

System Components

  • Slide Holder. The black aluminum Slide Holder consists of an outer frame that holds 2 to 40 microscope slides. The Slide Holder is moved manually along the Base Platform, thus allowing the microscope slides to either be placed in a Reagent or on top of a Wicking Pad. The simple IHC procedure alternates moving the Slide Holder between a Reagent and a Wicking Pad.
  • Base Platform. The black aluminum Base Platform holds the Nests in their proper positions.
  • Nests. The clear aluminum Nests are positioned onto the Base Platform to properly orient the Slide Holder, Reagent Trays, and Wicking Pads.
  • Reagent Trays. The Reagent Trays hold and separate each of the various IHC Reagents. The tissues on the microscope slides are incubated in a particular IHC Reagent, and then moved to a Wicking Pad.
  • Wicking Pads. The highly absorbent Wicking Pads capture the Reagents after they have been incubated with the tissue on the microscope slides.
  • Capillary Gap Slides, with Tissue Location. These special Capillary Gap microscope slides have a painted-raised label (and "end feet") on one side. The Tissue is placed on this side of the glass. When two Capillary Gap slides are placed label-to-label (face-to-face), the painted surfaces separate the tissues and form a Gap. When the Bottom of the slides are placed in a liquid (buffer, primary antibody, etc.), the liquid is gently drawn between the glass slides by Capillary Action and covers the tissue. The liquids are released onto a Wicking Pad by placing the Bottom of the slides onto the pad. The slides are Positively Charged ("+") to assist in tissue adherence to the glass surface.

Set Up

  1. Place the Base Platform of the IHC Select® Manual Staining System on a flat laboratory bench.
  2. Place a Wicking Pad in the second position from the left. Compress the corners of the pad to fit it snuggly in the Clear Aluminum Nest. Once it is snug, flatten the top so the surface of the pad is flat. This may be done by pressing in the center of the pad with your thumbs and gently lifting the corners. When properly positioned, the pad should fit snuggly in the Nest, and the top surface of the pad should be flat.
  3. The Reagent Trays are numbered 1-10 inside the bottom of the reagent wells. This Orientation will assist in Inserting the Reagent Trays into the Nests and in placing the Reagents in the correct position.
  4. Place the Reagent Trays into the Clear Aluminum Nests by inserting the Right Hand Side first (the side with the number ‘10' in the bottom of the reagent well). There is an extension on the right hand side that fits into a slot in the Nest.
  5. Snap the Reagent Trays into the Nests by pressing down on the left side of the Tray. An audible ‘Snap' is heard when it is correctly inserted. The left hand side of the tray also has an extension, and it is this extension that snaps into the Nest. When correctly inserted, the numbers in the bottom of the Tray should read 1-10 from the left to the right.
  6. Complete the initial setup by having the following positions filled:
    1. Position 1: Two Reagent Trays
    2. Position 2: One Pad
    3. Position 3: Two Reagent Trays
    4. Position 4: Two Reagent Trays
      To remove a Reagent Tray, simply press the Left side of the Tray in toward the center and lift the edge of the Tray up. The Tray may then be removed from the Nest.
  7. The Slide Holder is Oriented Front to Back, and Left to Right. ‹CHEMICON INTERNATIONAL‹and one thumb indent, on the top edge, orient the front. Four finger indents, on the top edge, orient the back. The numbering of the microscope slide positions 1–10 orient left to right. The Row ‹A‹ microscope slides are the front row and the Row 'B' slides are the back row. This Orientation is important for putting the Microscope Slides in their proper position, and in assuring the Slides are positioned into the correct Reagent in the Reagent Trays.
  8. Note that the Slide Holder will only fit into the Nests when the Front is facing the Front.

Inserting Slides into the Holder

  1. The Rows A and B, and Slide Positions 1-10, are marked on the front and back, both sides, and the bottom of the Slide Holder. Turn the Slide Holder upside down on a flat surface for loading the slides.
  2. Note: The Row A slides are in the back while the slide holder is upside down. When turned rightside-up, the Row A slides will be in the front (toward you).
  3. The Capillary Gap Slides are loaded into the Slide Holder as a Slide Pair.
    Note: Each slide has a Raised, Painted Surface (‘Label' and ‘End Feet').
    Note: When the slides are placed together, Labeltoward- Label (‘Face-to-Face'), the painted surfaces produce a ‘Capillary Gap' between the glass surfaces of the slides. Both slides may contain tissue samples (usually 4-5 microns thick) as the Capillary Gap is approximately 150 microns.
  4. Carefully insert the 'Label' end of the Slide Pair into the Slide Holder.
  5. Note: The slides slip into the notched openings (e.g., A1. A2, etc.)
    Note: Be careful not to push the slides side-to-side (towards the ''A''s) as this might break weak slides. Wiggling the slides towards the numbers may help during the insertion of the Slide Pairs.
  6. Continue to insert additional Slide Pairs into the Slide Holder.
  7. Align the bottom of the Slide Pairs in the Slide Holder by using the back (unpainted) side of a blank slide. All of the Bottom ends of the Slide Pairs should be aligned (‘flat').
  8. Note: Aligning the Slide Pairs is very important for proper wicking of the reagents from the Slide Pairs into the Wicking Pad.
  9. Align the edges of the Slide Pairs in the Slide Holder by using the back (unpainted) side of a blank slide. All of the edges of the Slide Pairs should be aligned (‘parallel').
Note: Aligning the Slide Pairs is very important for With the Slide Holder facing forward, place the Slide Holder into the first Nest.
Note: To assist in the ‘Forward' orientation, the following design elements have been incorporated into the Slide Holder:
  • The ‘CHEMICON INTERNATIONAL' etching on the top of the Slide Holder faces forward
  • The top of the Slide Holder has one indent in the front top for your thumb, and four indents in the top back for your fingers, and
  • The ‘Feet' of the Slide Holder can only be placed into the Nests with the Slide Holder facing forward.

IHC Protocol

IHC Protocol Outline (for additional information, and IHC reagents, see www.millipore.com to view the IHC Select® Detection Reagents Product Insert). The IHC Select® Primary Antibodies, Detection Reagents, and Ancillary Products have been optimized for staining formalin-fixed, paraffin-embedded tissues. Adjustments may be needed for other IHC staining (e.g., changing incubation times).

  1. Prior to beginning the removal of the paraffin from the tissue section on the slide (deparaffinization), bake the slides in a 60℃ oven for 1-2 hours. Allow the slide to come to room temperature before beginning deparaffinization.
  2. Deparaffinize by immersing the slides in Xylenes (4 x 5 min.), 100% Ethanol (2 x 2 min.), 70% Ethanol (2 x 2 min.), 30% Ethanol (2 x 2 min.), and Distilled or Deionized Water (2 x 2 min.).
  3. If the Target to detect (e.g., Vimentin) requires Tissue Pretreatment (i.e., Epitope Retrieval, sometimes called Antigen Retrieval) do it now. For example pretreat with Citrate Buffer pH 6.0 using a steamer, autoclave, pressure cooker, etc. (Battifora, et al., 1995). Allow the tissues to come to room temperature gradually after Epitope Retrieval (e.g., remove from steamer or pressure cooker, place at room temperature and leave to cool for at least 10 minutes).
  4. Note:While the slides are being Deparaffinized (or during Epitope Retrieval) set up the IHC Select® Manual Staining System.
  5. If Streptavidin-HRP, and DAB are used, incubate sections with 3% Hydrogen Peroxide for 10 minutes.
  6. Rinse sections with 1x Rinse Buffer (with T-20) for at least 1 minute.
  7. Put the Slides together to form Slide Pairs and load Slide Pairs into the IHC Select Slide Holder.
  8. Note:This can be done easily by placing fresh 1x Rinse Buffer (with T-20) in a Reagent Tray, and forming the slide pairs with fresh Buffer between the slides. Load the Slide Pair, with Buffer between the slides, into the Slide Holder. Do not let the tissue on the slides dry out at any time.
  9. Rinse sections with 1x Rinse Buffer (with T-20) for 5 x 30 seconds (use Wicking Pad between each rinse).
  10. Note:Do not leave the slides on the pad longer than 30 seconds or the tissue sections may dry out.
  11. Wick out on Pad.
  12. Incubate sections with Blocking Reagent (5 minutes).
  13. Wick out on Pad.
  14. Rinse sections with 1x Rinse Buffer (with T-20) for 1 x 30 seconds.
  15. Wick out on Pad.
  16. Incubate sections with Primary Antibody OR a Negative Control Reagent (if using a prediluted IHC Select® Primary Antibody, incubate for 10 minutes; antibodies other than IHC Select must be titered before deciding on a specific concentration and incubation time.
  17. Wick out on Pad.
  18. Rinse sections with 1x Rinse Buffer (with T-20) for 5 x 30 seconds (use Wicking Pad between each rinse).
  19. Wick out on Pad.
  20. Incubate sections with Biotinylated Secondary Antibody for 10 minutes.
  21. Wick out on Pad.
  22. Rinse sections with 1x Rinse Buffer (with T-20) for 5 x 30 seconds (use Wicking Pad between each rinse).
  23. Wick out on Pad.
  24. Incubate sections with Streptavidin-Enzyme Conjugate (for 10 minutes; for example SA-HRP).
  25. Wick out on Pad.
  26. Rinse sections with 1x Rinse Buffer (with T-20) for 2 x 30 seconds (use Wicking Pad between each rinse).
  27. Note: Change Wicking Pad at this time (replace with fresh pad).
  28. Rinse sections with 1x Rinse Buffer (with T-20) for 5 x 30 seconds (use Wicking Pad between each rinse).
  29. Wick out on Pad.
  30. Incubate sections with Chromogen (for 10 minutes; for example DAB).
  31. Wick out on Pad.
  32. Rinse sections with 1x Rinse Buffer (with T-20) for 5 x 30 seconds (use Wicking Pad between each rinse).
  33. Wick out on Pad.
  34. Incubate sections with Counter Stain (for 1 minute; for example Hematoxylin).
  35. Wick out on Pad.
  36. Rinse sections with 1x Rinse Buffer (with T-20) for 5 x 30 seconds (use Wicking Pad between each rinse).
  37. Wick out on Pad.
  38. Rinse sections with distilled or deionized water (hold here until next step).
  39. Cover slip using an aqueous mounting media, or dehydrate through a graded series of alcohols, immerse in Xylenes and cover slip using a permanent mounting media.
  40. View through a transmitted light microscope.
  41. See the IHC Select® Detection Reagents Product Insert for assistance in Quality Control and interpretation of results.

Reference

Battifora H, Alsabeh R, Jenkins KA, Gown AM: "Epitope Retrieval (Unmasking) in Immunohistochemsitry". Advances in Pathology and Laboratory Medicine, vol. 8, 101-118, 1995.

출처: http://www.millipore.com