Feulgen Staining Procedure
Feulgen Staining Procedure
1 Overview
1.1 Purpose and Scope
This procedure is used to establish a routine Feulgen staining workflow. It is applicable to the specific visualization of DNA in cultured cell coverslips, tissue sections, cytology smears, and chromosome preparations. It can be used for nuclear DNA localization, chromosome structure studies, and relative DNA content analysis.
1.2 Principle
(1) The Feulgen reaction is a classic DNA-specific staining method. After mild hydrochloric acid hydrolysis, purine bases in DNA molecules are dissociated, exposing free aldehyde groups at the C-1 position of deoxyribose.
(2) The leucofuchsin in Schiff reagent reacts with these aldehyde groups and reforms a magenta compound. Therefore, DNA-containing regions in cells show a magenta-positive reaction.
(3) This reaction has good specificity for DNA. It can clearly display cell nuclei and chromosome structures, and the approximate proportional relationship between staining intensity and DNA content can be used for relative quantitative analysis.
1.3 Experimental Quality Requirements
(1) Cell nuclei or chromosomes should show clear and stable magenta staining.
(2) Cytoplasmic background should be low, and nuclear structural boundaries should be clear.
(3) Staining intensity should be consistent within the same batch of specimens to facilitate intergroup comparison and relative quantitative analysis.
2 Materials and Reagents
2.1 Sample Types
(1) Cultured cell coverslip specimens.
(2) Paraffin tissue sections.
(3) Cytology smears.
(4) Chromosome spreads or squash preparations.
2.2 Main Materials
(1) HeLa human cervical cancer epithelial cells or other cells to be tested.
(2) Glass slides and coverslips.
(3) Fine-tip forceps, pipettes, and absorbent filter paper.
2.3 Main Reagents
(1) Hanks’ solution.
(2) 1 mol/L hydrochloric acid.
(3) Schiff reagent.
(4) Carnoy’s fixative, methanol:glacial acetic acid = 3:1.
(5) 0.5% Light Green.
(6) Distilled water.
(7) 70% ethanol.
(8) 80% ethanol.
(9) 95% ethanol.
(10) Absolute ethanol.
(11) Xylene.
(12) Neutral balsam.
2.4 Instruments and Equipment
(1) Standard light microscope.
(2) Constant-temperature water bath.
(3) CO₂ incubator.
(4) Staining jars.
3 Preparation of Staining Solutions and Samples
3.1 Preparation of Hanks’ Solution
(1) Stock solution A
Dissolve 160 g NaCl, 8 g KCl, 2 g MgSO₄·7H₂O, and 2 g MgCl₂·6H₂O in 800 mL distilled water. Separately dissolve 2.8 g anhydrous CaCl₂ in 100 mL distilled water. Mix the two solutions, add water to 1000 mL, filter, add 2 mL chloroform as preservative, and store at 4°C.
(2) Stock solution B
Dissolve 3.04 g Na₂HPO₄·12H₂O, 1.2 g KH₂PO₄, and 20.0 g glucose in 800 mL distilled water. Filter, add 80 mL of 0.5% phenol red, add water to 1000 mL, then add 2 mL chloroform as preservative. Store at 4°C.
(3) Working solution
Mix 1 part stock solution A with 1 part stock solution B and 18 parts double-distilled water. Dispense after mixing, autoclave, and store at 4°C. Before use, adjust to the required pH with sterile 5.6% NaHCO₃.
3.2 Preparation of Schiff Reagent
(1) Add 0.5 g basic fuchsin to 100 mL boiling distilled water. Continue boiling for 5 min and stir until fully dissolved.
(2) Cool to 50°C, filter into an amber bottle, and add 10 mL of 1 mol/L hydrochloric acid.
(3) Cool to 25°C, add 1 g anhydrous sodium bisulfite, shake thoroughly, and store protected from light overnight.
(4) The next day, add 0.25 g activated charcoal, shake vigorously for 1 min, and filter. The resulting solution should be completely colorless.
(5) Schiff reagent should be stored sealed, protected from light, and at low temperature. Bring it to room temperature before use.
3.3 Fixation Requirements
(1) Carnoy’s fixative generally gives good results in Feulgen staining and is suitable for DNA visualization in cells and tissues.
(2) For relative quantitative analysis, the fixative type and fixation time should be kept consistent to reduce pretreatment variation.
(3) After fixation, samples should be protected from repeated drying and contamination.
3.4 Control Setup
(1) An unhydrolyzed control is recommended for each staining batch.
(2) Control sections or coverslips should skip hydrochloric acid hydrolysis and go directly into Schiff reagent. In theory, they should show a negative or very weak reaction.
(3) The unhydrolyzed control should not remain in Schiff reagent for too long, usually around 0.5 h, to avoid false positivity caused by the acidity of the reagent itself.
4 Experimental Procedure
4.1 Cell Culture and Coverslip Preparation
(1) Seed HeLa cells onto coverslips for culture.
(2) Culture for 24–48 h until the cells form a monolayer.
(3) Mark the cell-facing side of the coverslip during operation to avoid damaging the cell layer during subsequent washing.
4.2 Washing and Fixation
(1) Take one cell coverslip and rinse three times with Hanks’ solution, pH 7.2–7.4.
(2) Gently absorb excess liquid with absorbent filter paper. Do not rub the cell surface directly.
(3) Place the coverslip in Carnoy’s fixative for 30 min.
(4) After fixation, wash three times with distilled water.
4.3 Mild Hydrochloric Acid Hydrolysis
(1) Place the coverslip in 1 mol/L hydrochloric acid.
(2) Hydrolyze in a 60°C constant-temperature water bath for 10 min.
(3) Immediately remove the coverslip after hydrolysis and wash once with distilled water to stop further reaction.
4.4 Schiff Reaction
(1) Transfer the coverslip into Schiff reagent.
(2) React in the dark for 30 min.
(3) During operation, minimize exposure of Schiff reagent to air and protect it from light.
4.5 Washing and Color Development
(1) After the Schiff reaction, rinse with running water for 5 min.
(2) Then wash three times with distilled water.
(3) At this stage, DNA-positive regions should gradually develop a stable magenta color.
4.6 Counterstaining
(1) Place the coverslip in 0.5% Light Green for 1–3 min.
(2) Counterstaining should not be too intense, otherwise the nuclear DNA signal may be obscured.
(3) If the main purpose is pure DNA localization, the counterstaining intensity can be optimized according to the system.
4.7 Dehydration, Clearing, and Mounting
(1) Dehydrate sequentially through 70% ethanol, 80% ethanol, 95% ethanol, and absolute ethanol.
(2) Immerse the coverslip in xylene for clearing for 5 min.
(3) Add one drop of neutral balsam onto a glass slide.
(4) Mount the coverslip with the cell-facing side down.
(5) Avoid air bubbles after mounting and ensure that the mounting medium is evenly distributed.
5 Reference Operation Sequence
HeLa cell coverslip culture for 24–48 h → rinse three times with Hanks’ solution → fix in Carnoy’s fixative for 30 min → wash three times with distilled water → hydrolyze in 1 mol/L hydrochloric acid at 60°C for 10 min → wash once with distilled water → react in Schiff reagent in the dark for 30 min → rinse with running water for 5 min → wash three times with distilled water → counterstain with 0.5% Light Green for 1–3 min → dehydrate through 70% ethanol → 80% ethanol → 95% ethanol → absolute ethanol → clear in xylene for 5 min → mount with neutral balsam → observe under microscope
6 Key Points for Result Interpretation
6.1 Cell Nuclei and Chromosomes
(1) DNA-rich regions in cell nuclei and chromosomes should appear magenta or reddish-purple.
(2) When nuclear staining is uniform, nuclear boundaries and chromatin distribution are clearer.
(3) In chromosome preparations, chromosome outlines and densely stained regions should be well displayed.
6.2 Cytoplasmic Background
(1) The cytoplasm generally does not show a typical Feulgen-positive reaction.
(2) When Light Green counterstaining is appropriate, the cytoplasm appears as a pale green background, helping highlight the nuclear DNA signal.
6.3 Control Samples
(1) The unhydrolyzed control should be negative or nearly negative.
(2) If the unhydrolyzed control shows an obvious magenta reaction, false positivity or inappropriate operating conditions should be considered first.
7 Quality Control Points
7.1 Fixation Quality Control
(1) Carnoy’s fixative is usually more suitable for the Feulgen reaction.
(2) Different fixatives affect reaction intensity and stability. The fixation system should be kept as consistent as possible within the same type of experiment.
(3) For quantitative analysis, the fixative and fixation time must be kept consistent.
7.2 Hydrolysis Condition Control
(1) Hydrolysis time and temperature are the most critical technical variables in this experiment.
(2) Insufficient hydrolysis results in inadequate exposure of free aldehyde groups and weakens the reaction.
(3) Excessive hydrolysis causes further DNA degradation and also weakens staining; in severe cases, a negative reaction may occur.
(4) Under routine conditions, hydrolysis time is generally controlled at 10–15 min and adjusted according to specimen type.
7.3 Schiff Reagent Control
(1) The quality of Schiff reagent directly determines the color development result.
(2) Schiff reagent is easily oxidized and turns red when exposed to air. Once the reagent fails, it should no longer be used.
(3) The same batch of Schiff reagent should be used as much as possible within the same experiment.
7.4 Coverslip Handling Control
(1) Since cultured cell coverslips are used in this experiment, the front and back sides of the coverslip should be clearly identified throughout the procedure.
(2) During running-water rinsing, avoid direct water impact on the cell surface to prevent detachment or damage to the cell layer.
8 Common Problems and Cause Analysis
8.1 Staining Too Light
(1) Insufficient hydrolysis.
(2) Schiff reagent activity has decreased or the reagent has failed.
(3) Improper fixation, especially when an unsuitable fixation system is used, may result in a weak reaction.
(4) Running-water color development time is insufficient.
8.2 Weakened Staining or False Negative
(1) Excessive hydrolysis.
(2) Hydrochloric acid temperature is too high or treatment time is too long.
(3) Further loss of deoxyribose reduces the ability to react with Schiff reagent.
8.3 High Background or False Positive
(1) The unhydrolyzed control remains in Schiff reagent for too long.
(2) Schiff reagent is oxidized or contaminated by air exposure.
(3) Washing is insufficient, leaving unbound dye residue.
8.4 Cell Layer Damage or Uneven Staining
(1) The front and back sides of the coverslip are not clearly identified, causing damage to the cell-facing surface during operation.
(2) Running water directly impacts the cell layer.
(3) Operation during fixation, washing, or dehydration is too rough.
9 Applications
9.1 DNA Localization
(1) Used to display the location and distribution of DNA in cell nuclei.
(2) Suitable for chromosome and nuclear structure studies.
9.2 Cytological and Histological Analysis
(1) Used for observing nuclear morphology and chromatin structure.
(2) Helps distinguish cells with different nuclear structures and differentiation states.
9.3 Relative DNA Quantitative Analysis
(1) Relative measurement can be performed based on the approximate proportional relationship between staining intensity and DNA content.
(2) Suitable for combined application with image analysis or microscopic quantitative methods.
10 Safety and Operating Standards
10.1 Personal Protection
(1) Wear a lab coat and disposable gloves during the experiment.
(2) Handle hydrochloric acid, Carnoy’s fixative, xylene, and Schiff reagent in a well-ventilated environment.
10.2 Waste Disposal
(1) Waste liquids containing hydrochloric acid, Schiff reagent, ethanol, and xylene should be collected separately by category.
(2) All waste liquids and contaminated consumables should be disposed of according to laboratory chemical waste handling regulations.
11 Selection of Key Reagents and Materials for Feulgen Staining
Cat. No. | Product Name | Grade and Purity | Corresponding Step | Use |
Hanks′ Balanced Salt solution | for cell culture, liquid,With sodium bicarbonate,sterile-filtered | Rinsing | Corresponds to the Hanks’ solution rinsing step before HeLa coverslip staining | |
Hanks' Balanced Salt Solution (with Ca2+ & Mg2+) |
| Rinsing | Can be used as a ready-to-use balanced salt solution option in the Hanks’ solution system | |
Carnoy's Fluid | BioReagent, ready-to-use | Fixation | Corresponds to the recommended fixation system for the Feulgen reaction | |
Carnoy Fixative Ⅱ | BioReagent, ready-to-use | Fixation | Can be used as an alternative Carnoy fixation system | |
Schiff Reagent | BioReagent,Biological Stain,for microscopy | Schiff reaction | Corresponds to the core color development step in the Feulgen reaction | |
Schiff′s reagent for aldehydes | for microscopy | Schiff reaction | Can be used in aldehyde color development reaction systems | |
Water | for biotechnology nuclease-free, sterile | Rinsing / solution preparation | Can be used as standardized water in cytology experiments | |
Bright green staining solution (1%) | BioReagent,Biological Stain,for microscopy,1% | Counterstaining | Can be diluted for use in a 0.5% Light Green counterstaining system | |
Brilliant green | AR | Counterstain preparation | Suitable for preparing Light Green counterstaining solution | |
Brilliant green | High-purity, ≥95% | Counterstain preparation | Suitable for preparing Light Green counterstaining solution | |
Xylene | Premium-Grade Reagents, ≥99%, xylene isomer and ethyl benzene | Clearing | Corresponds to the xylene clearing step | |
Xylene | ACS, ≥98.5%, isomers plus ethylbenzene | Clearing | Suitable for standardized clearing treatment | |
Xylene | Anhydrous Grade, ≥98%, mixture of isomers | Clearing | Corresponds to routine clearing treatment | |
Neutral gum | FMP | Mounting | Corresponds to the neutral balsam mounting step |