Gill's Hematoxylin #3 is a triple-strength progressive hematoxylin formulation for nuclear staining in routine histology, including hematoxylin and eosin (H&E) procedures. It is the strongest of the three Gill hematoxylin formulations and is used at full strength without routine mixing or dilution.
In progressive staining, nuclear intensity is established by controlling hematoxylin exposure time rather than by deliberately overstaining followed by acid differentiation. Gill's #3 provides greater nuclear staining intensity with shorter staining times than the lower-strength Gill formulations. Chromatin is expected to stain blue to blue-black after appropriate staining and bluing.
Key Properties
- Product number: 24244
- Stain type: Progressive nuclear stain
- Formulation: Gill's Hematoxylin #3, triple strength
- Primary application: Histology
- Use: Hematoxylin and eosin (H&E) staining
- Format: Liquid; use at full strength
- Mordant: Aluminum sulfate
- Oxidizing agent: Sodium iodate
- Contains acetic acid for pH control
- Contains ethylene glycol to reduce surface precipitate formation and stabilize staining performance
- Expected chromatin staining: Blue to blue-black
- Available sizes: 500mL and 1000mL
- Storage: Room temperature
Applications
Routine H&E Histology
Gill's Hematoxylin #3 is used as the nuclear stain in routine H&E procedures. After hematoxylin staining and bluing, eosin is applied as the counterstain for cytoplasmic and extracellular tissue components.
Paraffin-Embedded Tissue Sections
Gill's #3 is used for progressive nuclear staining of deparaffinized tissue sections. Polysciences has evaluated the formulation in H&E protocols using formalin-fixed, paraffin-embedded tissues.
Frozen Sections
Gill's #3 may also be used in H&E staining of frozen tissue sections. Staining time should be established for the fixation method, tissue type, section thickness, and staining sequence.
Automated H&E Staining
Gill's #3 has been evaluated in progressive H&E protocols on automated slide stainers. Hematoxylin exposure time and subsequent staining steps should be validated for the specific instrument, tissue preparation, section thickness, and laboratory protocol.
Staining Characteristics & Method Considerations
- Gill's #3 is a progressive hematoxylin formulation
- Nuclear staining intensity is controlled by hematoxylin exposure time
- Routine acid-alcohol differentiation is not part of the standard progressive Gill method
- A bluing step follows hematoxylin staining in standard H&E workflows
- Gill's #3 is the strongest formulation in the Gill series and is intended for shorter staining times than Gill's #1 or #2 when comparable nuclear intensity is required
- Tissue type, fixation, section thickness, staining time, reagent condition, and bluing conditions can affect the final appearance of nuclear staining
Progressive Gill staining differs from regressive hematoxylin methods. In a progressive method, staining is stopped when the desired nuclear intensity is reached. Regressive methods intentionally over-stain and then use an acid differentiation step to remove excess hematoxylin.
Application Tips
- Use Gill's Hematoxylin #3 at full strength; routine dilution or additional preparation is not required
- Establish hematoxylin exposure time for the tissue type, fixation method, section thickness, and staining platform
- Use a bluing step after hematoxylin staining before eosin counterstaining
- Do not add an acid-alcohol differentiation step unless it is part of a specifically validated laboratory procedure
- Maintain consistent staining time, section thickness, bluing conditions, and eosin exposure when comparing preparations
- Monitor staining performance with a representative control slide processed under consistent conditions
Handling & Storage
- Store at room temperature in the dark
- Keep the container tightly closed when not in use
- Minimize prolonged exposure of the staining solution to air
- Limit headspace in storage containers when practical to reduce oxidation during storage
- Evaluate staining performance microscopically if the reagent develops an unexpected color change or surface precipitate during extended storage or use
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FAQ
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1. What is Gill's Hematoxylin #3 used for?
Gill's Hematoxylin #3 is a progressive nuclear stain used primarily in routine histology, including hematoxylin and eosin staining of tissue sections.
2. What does triple strength mean for Gill's Hematoxylin #3?
Triple strength is the formulation designation for Gill's #3, the strongest of the three Gill hematoxylin formulations. It provides greater nuclear staining intensity with shorter staining times than the lower-strength Gill formulations.
3. Is Gill's Hematoxylin #3 a progressive or regressive stain?
Gill's #3 is a progressive hematoxylin. Staining is stopped when the desired nuclear intensity has been reached rather than intentionally overstaining the section and subsequently differentiating it with acid.
4. Does Gill's Hematoxylin #3 require acid differentiation?
Routine acid differentiation is not part of the standard progressive Gill staining method. An acid differentiation step is characteristic of regressive hematoxylin protocols.
5. Does Gill's Hematoxylin #3 need to be diluted before use?
No. Gill's Hematoxylin #3 is used at full strength. Routine mixing, dilution, or additional treatment is not required before use.
6. What color should chromatin appear after Gill's Hematoxylin #3 staining?
Chromatin is expected to appear blue to blue-black after appropriate hematoxylin staining and bluing.
7. What is the difference between Gill's Hematoxylin #2 and #3?
Gill's #2 is a double-strength progressive formulation used in histology, cytology, and immunohistochemical counterstaining. Gill's #3 is the stronger triple-strength formulation intended primarily for histology. See Gill's Hematoxylin #2.
8. Can Gill's Hematoxylin #3 be used on automated slide stainers?
Yes. Gill's #3 has been evaluated in progressive H&E protocols using automated slide stainers. Staining times and subsequent processing steps should be validated for the specific instrument and laboratory protocol.
9. What counterstain is used with Gill's Hematoxylin #3 in H&E staining?
Eosin is used as the counterstain in H&E procedures. Polysciences offers Eosin Y, 0.5% alcoholic solution, acidic for hematoxylin and eosin staining.
10. Is a bluing step used after Gill's Hematoxylin #3?
Yes. Bluing follows hematoxylin staining in standard H&E workflows. Scott's Bluing Reagent is one available reagent for this step.
11. How should Gill's Hematoxylin #3 be stored?
Store at room temperature in the dark in a tightly closed container. Limiting prolonged exposure to air helps reduce oxidation of the staining solution.
12. What sizes are available?
Gill's Hematoxylin #3 is available in 500mL and 1000mL sizes.
Safety & Documentation
Refer to Technical Data Sheet #192 for Gill hematoxylin formulation, use, storage, and reagent-condition guidance. See Technical Data Sheet #776 for manual and automated H&E staining protocols and the Safety Data Sheet for handling and safety information.
