# Detection of Carrageenanen in Cheese Using
## 3 Results and Discussion  
### Choice of Fixative Solution  
Fixation is one of the key steps in the preparation of samples for histological and immunohistochemical methods, and it has a significant effect on the quality of the whole method and the results obtained. Samples should be prepared to preserve their structure [20]. Fixation prevents tissue decomposition and also minimizes damage during other processes, such as dehydration, watering, and sample cutting [21]. It has been found that formaldehyde fixation and histological processing can lead to denaturation and epitope masking [22]. Immunoreactivity may be directly proportional to the storage of slides [23]. For some matrices, sample processing is already a routine practice. In particular, the histology of animal tissues [24; 25] and plant tissues [25; 26; 27]. Several studies are also available for foodstuffs of meat origin describing the method of their fixation [28; 29]. Processing for microscopic methods has also been described for cheese [9; 30; 31]. During fixation, the sample is intensively cross-linked and its structure is stabilized [32; 33; 34]; therefore, the hardness parameter of the sample was chosen as an indicator of suitable fixation. Extensive cross-linking can lead to loss or reduction of enzymatic activities [35]. The differences in firmness between the individual samples are shown in Figure 2.  
Figure 2: Comparison of texture measurement (firmness) results for processed cheese samples [g]. Effect of fixation to the firmness of processed cheese samples. Notes: 0—no fixation, 1—AFA, 2—10% formal, 3—100% ethanol, 4—[PERSON]’s solution.  
When comparing the texture of the fixed samples with the control sample that was not fixed (0_a--58.47 g and 0_b--37.53 g), the samples fixed in [PERSON]'s solution (4_a --164.43 g and 4_b--266.03 g) and in 10% formed (2_a--147.92 g and 2_b--36.98 g) had a similar consistency. The consistency of these samples was very soft and non-compact. Formalin can cause tissue swelling and can give cells a vitreous appearance [36]. In contrast, the highest texture values were recorded for samples that were fixed with ethanol (3_a--3087.34 g and 3_b--2257.79 g). The consistency of these samples was friable. The reason for this result is probably the dissolved lipids. Alcohol-based fixing solutions penetrate tissue poorly, precipitate proteins and dissolve lipids. Long-term fixation in absolute alcohol may lead to tissue shrinkage [37, 38]. High hardness and fragility can cause tissue damage during following sample manipulations [21]. [PERSON]'s solution also contains alcohol. However, the chloroform and acetic acid, which are in this solution too, prevent the shrinking of the tissue caused by ethanol [32]. The results show that the fixations (10% formed, ethanol and Carnoy's solution) are not suitable for processing of processed cheese samples.  
The graph shows that samples fixed in AFA fixative solution (1_a--1805.38 g and 1_b--645.65 g) have the most suitable consistency because these samples were not soft and remained compact. A suitable fixation has to strengthen and make it hard enough to minimize the damage and reduce the deformation during dehydration, embedding, and cutting [21]. The differences in values between the samples are most likely given by composition. For example, hydrocolloids have a significant impact on the texture profile, such as hardness, cohesion, springiness, chewing, and gumminess [39].  
A highly significant difference (\(p<0.01\)) was found between the individual samples of fixative solutions in comparison with the control sample except for the sample fixed in 10% formalin (\(p>0.05\)).